Derivatives of laspartomycin and preparation and use thereof

ABSTRACT

The present invention provides laspartomycin core peptides, laspartomycin core peptide derivatives, antimicrobial laspartomycin derivatives, methods for making laspartomycin core peptides, methods for making laspartomycin core peptide derivatives, methods for making antimicrobial laspartomycin derivatives, pharmaceutical compositions of antimicrobial laspartomycin derivatives, methods of inhibiting microbial growth and methods for treating and/or preventing microbial infections in a subject.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/760,328, filed Jan. 12, 2001, which claimed thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/219,095, filed Jul. 17, 2000 and U.S. Provisional Application No.60/220,950, filed Jul. 26, 2000. The above applications are incorporatedherein by reference in their entirety.

1. FIELD OF THE INVENTION

[0002] The present invention relates generally to antibiotics andantimicrobial derivatives. More particularly, the present inventionrelates to intermediates usefuil for synthesizing laspartomycinderivatives as well as the laspartomycin derivatives.

2. BACKGROUND OF THE INVENTION

[0003] Laspartomycin (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawaet al., 1968, J. Antibiot., 21, 55; Naganawa et al., 1970, J. Antibiot.,23, 423 which are herein incorporated by reference) is closely relatedto antibiotics such as zaomycin (Kuroya, 1960, Antibiotics Ann., 194;Kuroya, Japanese Patent No. 8150), crystalomycin (Gauze et al., 1957,Antibiotiki, 2, 9), aspartocin (Shay et a., 1960, Antibiotics Annual,194; Hausman et al., 1964, Antimicrob. Ag. Chemother., 352;

[0004] Hausman et al., 1969, J. Antibiot., 22, 207; Martin et al., 1960,J. Am. Chem. Soc., 2079), amphomycin (Bodanszky et. al., 1973, J. Am.Chem. Soc., 95, 2352), glumamycin (Fujino et al., 1965, Bull. Chem. Soc.Jap., 38, 515), daptomycin (Debono et. al., 1988, J. Antibiotics, 41,1093), Antibiotic A-1437 (Hammann et. al., EP 0 629 636 B1; Lattrell etal., U.S. Pat. No. 5,629,288), Antibiotic A54145 (Fukada et al., U.S.Pat. No. 5,039,789; Boeck et al., 1990, J. Antibiotics, 43, 587), andtsushimycin (Shoji et. al., 1968, J. Antibiot., 21, 439). The abovecompounds are lipopeptide antibiotics which typically inhibit grampositive bacteria. Generally, lipopeptide antibiotics consist of eithera cyclic core peptide or a cyclic core depsipeptide acylated with alipophilic fragment such as an unsaturated fatty acid.

[0005] Laspartomycin, produced by fermenting the microorganismStreptomyces viridochromogenes var. komabensis, was first isolated whilescreening for compounds active against resistant staphylococci (Naganawaet al., 1968, J. Antibiot., 21, 55; Umezawa et al., U.S. Pat. No.3,639,582). Laspartomycin was characterized by conventional methods andwas shown to be active against a variety of gram positive bacteria,including staphylococci and some fungi (id.). Elemental analysis andamino acid analysis provided a molecular weight of about 1827 for thelipopeptide antibiotic, while amino acid analysis indicated the presenceof the amino acids threonine and diaminobutryic acid in the peptideportion of laspartomycin (id.).

[0006] In other studies, the major lipophilic fragment of laspartomycinwas shown to be trans-2-isopentadecanoic acid 2, illustrated below(Naganawa et al., 1970, J. Antibiot. 23, 423). In contrast, thelipophilic portions of antibiotics such as aspartocin (Hausmann et al.,1963, Antimicr. Agents & Chemoth., 352, 1962), glumamycin (Inoue, 1962,Bull. Chem. Soc. Jap., 35, 1255), tsushimycin (Shoji et al., 1968, J.Antibiot., 21, 439) and amphomycin (Shoji et al., 1969, J. Antibiot.,22, 473) are all derived from cis β-γ unsaturated carboxylic acids.

[0007] The results described in the instant Application indicate thatthe amino acid analysis and the molecular weight disclosed in the artare incorrect (Umezawa et al., U.S. Pat. No. 3,639,582; Naganawa et al.,1968, J. Antibiot., 21, 55). In particular current studies disclosed inthis Application show that the peptide core of laspartomycin containsnovel amino acids not found in other known lipopeptide antibacterialantibiotics. For example, laspartomycin is the only member of theantibacterial lipopeptide family that contains diaminopropionic acid inthe peptide core. Amphomycin, aspartocin, zaomycin, tsushimycin, andantibiotic A-1437 contain, instead, 2,3-diaminobutyric acid in thepeptide portion of the molecule (Kuroya, 1960, Antibiotics Ann., 194;Gauze et al.,1957, Antibiotiki, 2, 9; Shay et al., 1960, AntibioticsAnnual, 194-198; Hausman et al., 1964, Antimicrob. Ag. Chemother., 352;Hausman et al., 1969, J. Antibiot., 22, 207; Martin et al., 1960, J. Am.Chem. Soc., 2079; Bodanszky et. al., 1973, J. Am. Chem. Soc., 95, 2352;Fujino et al., 1965, Bull. Chem. Soc. Jap., 38, 515; Hammann et. al., EP0 629 636 B1; Lattrell et al., U.S. Pat. No. 5,629,288; Shoji et al.,1968, J. Antibiot., 21, 439). Additionally, laspartomycin containsallo-threonine, which is not found in the other known lipopeptides.Further laspartomycin is the smallest of the known lipopeptides with amolecular weight of about 1247 for the cyclic core peptide acylated withcompound 2.

[0008] Despite the efficacy of laspartomycin against gram positivebacteria, the medicinal chemistry of this lipopeptide antibacterialantibiotic has remained largely unexplored. However, given the recentdramatic rise of antibiotic-resistant pathogens and infectious diseases,caused in part, by frequent over use of antibiotics, the need for newantimicrobial agents is urgent (Cohen et al., 1992, Science, 257,1050-1055). Specifically, methicillin resistant bacteria are aparticular problem since they are also resistant to a wide variety ofantibiotics other than methicillin (Yoshida et al., U.S. Pat. No.5,171,836). Gram positive bacteria, such as Staphylococci, which causepersistent infections, are especially dangerous when methicillinresistant. Even more alarmingly, strains of Enterococcus faecium thatare resistant to vancomycin have been recently observed (Moellering,1990, Clin. Microbiol. Rev., 3, 46). Strains resistant to vancomycinpose a serious health threat to society since vancomycin is theantibiotic of last resort for several harmful pathogens. Thus, there isa general need for antibiotic agents and a specific need for antibioticagents that are active against microbes resistant to methicillin orvancomycin.

3. SUMMARY OF THE INVENTION

[0009] The present invention addresses this and other needs in the artby providing antimicrobial laspartomycin derivatives, pharmaceuticalcompositions of antimicrobial laspartomycin derivatives, methods formaking antimicrobial laspartomycin derivatives, methods for inhibitingmicrobial growth and methods for treating or preventing microbialinfections in a subject. The present invention also provides alaspartomycin core peptide, methods for making the laspartomycin corepeptide and a laspartomycin core peptide derivative and methods formaking the laspartomycin core peptide derivative all of which are alluseful in synthesizing antimicrobial laspartomycin derivatives.

[0010] In one aspect, the present invention provides a laspartomycincore peptide derivative that may be used as a key intermediate in thesynthesis of antimicrobial laspartomycin derivatives. An essential partof the laspartomycin core peptide derivative is a core cyclic peptideattached to a nitrogen atom which may be part of a variety of functionalgroups such as, for example, a carbamate, amide or sulfonamide.

[0011] In one embodiment, the laspartomycin core peptide derivativeincludes a linker which is typically attached to the nitrogen of thelaspartomycin core peptide. The linker may be derived from compoundssuch as amino acids, polyamides, polyamines, polyethers,polysulfonamides or other linkers known to those of skill in the art.The linker typically includes a linking group which may be any chemicalfunctionality that can participate in covalent bond formation. Thelinking group provides a site for further modification of thelaspartomycin core peptide derivative. For example, the linking groupmay be modified with a lipophilic moiety to provide a laspartomycinderivative of the invention.

[0012] Thus, in one illustrative embodiment, the present inventionprovides a laspartomycin core peptide derivative according to structuralformula (I):

Y ¹—L—X¹—N(R¹)—R  (I)

[0013] or a salt or hydrate thereof, wherein either:

[0014] (i) Y¹—L—X¹ taken together is hydrogen; or

[0015] (ii) Y¹ is a linking group;

[0016] L is a linker;

[0017] X¹ is selected from the group consisting of —CO—, —SO₂—, —CS—,—PO—, —OPO—, —OC(O)—, —NHCO— and —NR¹CO—;

[0018] N is nitrogen;

[0019] R¹ is selected from the group consisting of hydrogen, (C₁-C₁₀)alkyl optionally substituted with one or more of the same or differentR² groups, (C₁-C₁₀) heteroalkyl optionally substituted with one or moreof the same or different R² groups, (C₅-C₁₀) aryl optionally substitutedwith one or more of the same or different R² groups, (C₅-C₁₅) arylaryloptionally substituted with one or more of the same or different R²groups, (C₅-C₁₅) biaryl optionally substituted with one or more of thesame or different R² groups, five to ten membered heteroaryl optionallysubstituted with one or more of the same or different R² groups,(C₆-C₁₆) arylalkyl optionally substituted with one or more of the sameor different R² groups and six to sixteen membered heteroarylalkyloptionally substituted with one or more of the same or different R²groups;

[0020] each R² is independently selected from the group consisting of—OR³, —SR³, —NR³R³, —CN, —NO₂, —N₃, —C(O)OR³, —C(O)NR³R³, —C(S)NR³R³,—C(NR³)NR³R³, —CHO, —R³CO, —SO₂R³, —SOR³, —PO(OR³)₂, —PO(OR³), —CO₂H,—SO₃H, —PO₃H, halogen and trihalomethyl;

[0021] each R³ is independently selected from the group consisting ofhydrogen, (C₁-C₆) alkyl, (C₅-C₁₀) aryl, 5-10 membered heteroaryl,(C₆-C₁₆) arylalkyl and six to sixteen membered heteroarylalkyl; and

[0022] R is the core cyclic peptide of laspartomycin.

[0023] In another aspect, the present invention provides antimicrobiallaspartomycin derivatives. The antimicrobial laspartomycin derivativesare generally laspartomycin core peptide derivatives of the inventionthat have been further modified with a lipophilic moiety. The lipophilicmoiety will usually be attached to a linking group covalently bonded tothe nitrogen atom of the core peptide derivative.

[0024] Thus, in another illustrative embodiment, the present inventionprovides an antimicrobial laspartomycin derivative according tostructural formula (II):

Y ²—(X²—X³)—(L)—(X¹—N(R¹)—R  (II)

[0025] or a pharmaceutically acceptable salt or hydrate thereof,wherein:

[0026] Y² is a lipophilic group;

[0027] X¹ is selected from the group consisting of —CO, —SO₂—, —CS—,—PO—, —OPO—, —OC(O)—, —NHCO— and —NR¹CO—;

[0028] X² is a linked group;

[0029] X³ is a linked group; and

[0030] N, L, R¹ and R are as previously defined for Formula (I).

[0031] In a third aspect, the present invention provides a method formaking a laspartomycin core peptide that includes culturing themicroorganism Streptomyces viridochromogenes, ssp. komabensis (ATCC29814) in a culture medium to provide laspartomycin. Isolation oflaspartomycin followed by cleavage of a lipophilic fragment provides thelaspartomycin core peptide.

[0032] In a fourth aspect, the present invention provides methods forsynthesizing a laspartomycin core peptide derivative. A linking moietymay be covalently attached to a laspartomycin core peptide to provide alaspartomycin core peptide derivative.

[0033] In a fifth aspect, the present invention provides approaches forsynthesizing antimicrobial laspartomycin derivatives. In a first method,a linking moiety may be covalently attached to a laspartomycin corepeptide to yield a laspartomycin core peptide derivative. Then, alipophilic group may be covalently attached to the laspartomycin corepeptide derivative to provide an antimicrobial laspartomycin derivative.In a second method, a linking moiety may be covalently attached to alipophilic group to yield a linker-lipophilic group. Then thelinker-lipophilic group may be covalently attached to the laspartomycincore peptide to provide an antimicrobial laspartomycin derivative.

[0034] In a sixth aspect, the present invention provides pharmaceuticalcompositions comprising the antimicrobial laspartomycin derivatives ofthe invention.

[0035] The pharmaceutical compositions generally comprise one or moreantimicrobial laspartomycin derivatives of the invention, and/orpharmaceutically acceptable salts thereof and a pharmaceuticallyacceptable carrier, excipient or diluent. The choice of carrier,excipient or diluent will depend upon, among other factors, the desiredmode of administration.

[0036] In a seventh aspect, the present invention provides methods ofinhibiting the growth of microbes such as gram positive bacteria,particularly, methicillin resistant Staphylococcus aureus and vancomycinresistant enterococci. The method generally involves contacting amicrobe with one or more antimicrobial laspartomycin derivatives of theinvention (or a pharmaceutically-acceptable salt thereof) in an amounteffective to inhibit the growth of the microbe. The method may bepractical to achieve a bacteriostatic effect, where the growth of themicrobe is inhibited, or to achieve a bactericidal effect, where themicrobe is killed.

[0037] In a final aspect, the present invention provides methods fortreating and/or preventing microbial infections in a subject such ashuman, plant or animal. The methods generally involve administering to asubject one or more of the antimicrobial laspartomycin derivatives orpharmaceutical compositions of the invention in an amount effective totreat or prevent a microbial infection in the human, animal or plant.The antimicrobial laspartomycin derivatives or pharmaceuticalcompositions may be administered systemically or applied topically,depending on the nature of the microbial infection.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions

[0038] As used herein, the following terms are intended to have thefollowing meanings.

[0039] “Laspartomycin:” refers to a mixture of at least three differentcompounds produced by culturing the microorganism Streptomycesviridochromogenes, ssp. komabensis (ATCC 29814) in a culture medium. Itshould be understood that the structure of the lipophilic side chain isdifferent in the three compounds.

[0040] The major component of laspartomycin (typically around 80% underthe fermentation and processing conditions used in this Application) isacylated with the C-15 α-β unsaturated carboxylic acid 2 shown above toprovide C-15 laspartomycin 4 shown below.

[0041] The two minor components are the C-14 and C-16 analogues of theC-15 α-β unsaturated carboxylic acid 2 shown above. The formulation ofthe culture medium and the ratio of the medium constituents has a directeffect on the ratio of the components of laspartomycin. Thus, noparticular component composition is intended by the use of the term“laspartomycin.”

[0042] “Lipophilic fragment:” refers to any lipophilic moiety attachedto the laspartomycin core peptide that is produced by culturing themicroorganism Streptomyces viridochromogenes, ssp. komabensis (ATCC29814) in a culture medium. Thus, lipophilic fragments include but arenot limited to, the C-14, C-15 and C-16 acyl analogues of the C-14, C-15and C-16 α-β unsaturated carboxylic acids described above.

[0043] “Core cyclic peptide:” refers to the cyclic peptide portion oflaspartomycin R shown below:

[0044] The dashed line indicates the carbon atom which is bonded tonitrogen in Formulas (I), (II) and (III).

[0045] “Laspartomycin core peptide:” refers to the peptide portion oflaspartomycin after cleavage of at least the lipophilic fragment. Thelaspartomycin core peptide may be represented by Formula (III) shownbelow:

R ^(X)NHR  (III)

[0046] where R^(X) is either H or NH₂CH(CH₂CO₂H)CO— and R is the corecyclic peptide of laspartomycin as defined above.

[0047] “Alkyl” refers to a saturated or unsaturated, branched,straight-chain or cyclic monovalent hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane, alkene or alkyne. Typical alkyl groups include, but are notlimited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propylssuch as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl,prop-1-en-2-yl, prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl;cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls suchas butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl,cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

[0048] The term “alkyl” is specifically intended to include groupshaving any degree or level of saturation, i.e., groups havingexclusively single carbon-carbon bonds, groups having one or more doublecarbon-carbon bonds, groups having one or more triple carbon-carbonbonds and groups having mixtures of single, double and triplecarbon-carbon bonds. Where a specific level of saturation is intended,the expressions “alkanyl,” “alkenyl,” and “alkynyl” are used. Theexpression “lower alkyl” refers to alkyl groups comprising from 1 to 8carbon atoms.

[0049] “Alkanyl” refers to a saturated branched, straight-chain orcyclic alkyl group. Typical alkanyl groups include, but are not limitedto, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.

[0050] “Alkenyl” refers to an unsaturated branched, straight-chain orcyclic alkyl group having at least one carbon-carbon double bond derivedby the removal of one hydrogen atom from a single carbon atom of aparent alkene. The group may be in either the cis or trans conformationabout the double bond(s). Typical alkenyl groups include, but are notlimited to, ethenyl; propenyls such as prop-1-en-1-yl , prop-1-en-2-yl,prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.; and the like.

[0051] “Alkynyl” refers to an unsaturated branched, straight-chain orcyclic alkyl group having at least one carbon-carbon triple bond derivedby the removal of one hydrogen atom from a single carbon atom of aparent alkyne. Typical alkynyl groups include, but are not limited to,ethynyl; propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.;butynyls such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl , etc.; andthe like.

[0052] “Aryl” refers to a monovalent aromatic hydrocarbon group derivedby the removal of one hydrogen atom from a single carbon atom of aparent aromatic ring system. Typical aryl groups include, but are notlimited to, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. In preferredembodiments, the aryl group is (C₅-C₁₄) aryl, with (C₅-C₁₀) being evenmore preferred.

[0053] “Arylaryl:” refers to a monovalent hydrocarbon group derived bythe removal of one hydrogen atom from a single carbon atom of a ringsystem in which two or more identical or non-identical parent aromaticring systems are joined directly together by a single bond, where thenumber of such direct ring junctions is one less than the number ofparent aromatic ring systems involved. Typical arylaryl groups include,but are not limited to, biphenyl, triphenyl, phenyl-naphthyl,binaphthyl, biphenyl-naphthyl, and the like. Where the number of carbonatoms in an arylaryl group are specified, the numbers refer to thecarbon atoms comprising each parent aromatic ring. For example, (C₅-C₁₄)arylaryl is an arylaryl group in which each aromatic ring comprises from5 to 14 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl,etc. Preferably, each parent aromatic ring system of an arylaryl groupis independently a (C₅-C₁₄) aromatic, more preferably a (C₅-C₁₀)aromatic. Also preferred are arylaryl groups in which all of the parentaromatic ring systems are identical, e.g., biphenyl, triphenyl,binaphthyl, trinaphthyl, etc.

[0054] “Biaryl:” refers to an arylaryl group having two identical parentaromatic systems joined directly together by a single bond. Typicalbiaryl groups include, but are not limited to, biphenyl, binaphthyl,bianthracyl, and the like. Preferably, the aromatic ring systems are(C₅-C₁₄) aromatic rings, more preferably (C₅-C₁₀) aromatic rings. Aparticularly preferred biaryl group is biphenyl.

[0055] “Arylalkyl” refers to an acyclic alkyl group in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl group. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylakenyl and/or arylalkynyl is used. In preferredembodiments, the arylalkyl group is (C₆-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₆) andthe aryl moiety is (C₅-C₁₄). In particularly preferred embodiments thearylalkyl group is (C₆-C₁₃), e.g., the alkanyl, alkenyl or alkynylmoiety of the arylalkyl group is (C₁-C₃) and the aryl moiety is(C₅-C₁₀).

[0056] “Heteroaryl” refers to a monovalent heteroaromatic group derivedby the removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, arsindole, carbazole,β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole,indole, indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like. In preferred embodiments,the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 memberedheteroaryl being particularly preferred. The most preferred heteroarylgroups are those derived from thiophene, pyrrole, benzothiophene,benzofuran, indole, pyridine, quinoline, imidazole, oxazole andpyrazine.

[0057] “Heteroarylalkyl” refers to an acyclic alkyl group in which oneof the hydrogen atoms bonded to a carbon atom, typically a terminal orsp³ carbon atom, is replaced with a heteroaryl group. Where specificalkyl moieties are intended, the nomenclature heteroarylalkanyl,heteroarylakenyl and/or heterorylalkynyl is used. In preferredembodiments, the heteroarylalkyl group is a 6-20 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of theheteroarylalkyl is 1-6 membered and the heteroaryl moiety is a5-14-membered heteroaryl. In particularly preferred embodiments, theheteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety is 1-3 membered and the heteroaryl moiety is a5-10 membered heteroaryl.

[0058] “Substituted:” refers to a group in which one or more hydrogenatoms are each independently replaced with the same or differentsubstituent(s). Typical substituents include, but are not limited to,—X, —R⁶, —O³¹ —, ═O, —OR, —SR⁶, —S⁻, ═S, —NR⁶R⁶, ═NR⁶, —CX₃, —CF₃, —CN,—OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O³¹ , —S(O)₂OH, —S(O)₂R⁶,—OS(O₂)O⁻, —OS(O)₂OH, —OS(O⁻)₂R⁶, —P(O)(O⁻)₂, —P(O)(OH)(O⁻),—OP(O)₂(O⁻), —C(O)R⁶, —C(S)R⁶, —C(O)OR⁶, —C(O)O⁻, —C(S)OR⁶, and—C(NR⁶)NR⁶R⁶, where each X is independently a halogen; each R⁶ isindependently hydrogen, halogen, alkyl, aryl, arylalkyl, arylaryl,arylheteroalkyl, heteroaryl, heteroarylalkyl —NR⁷R⁷, —C(O)R⁷ or—S(O)₂R⁷; and each R⁷ is independently hydrogen, alkyl, alkanyl,alkynyl, aryl, arylalkyl, arylheteralkyl, arylaryl, heteroaryl orheteroarylalkyl.

[0059] Reference will now be made in detail to preferred embodiments ofthe invention. While the invention will be described in conjunction withpreferred embodiments, it should be understood that it is not intendedto limit the invention to this preferred embodiment. To the contrary, itis intended to cover alternatives, modifications, and equivalents as maybe included within the spirit and scope of the invention as defined bythe appended claims.

4.2 The Invention

[0060] The present invention provides a laspartomycin core peptide,laspartomycin core peptide derivatives, antimicrobial laspartomycinderivatives, methods for making the laspartomycin core peptide, methodsfor making laspartomycin core peptide derivatives, methods for makingantimicrobial laspartomycin derivatives, pharmaceutical compositions ofantimicrobial laspartomycin derivatives, methods of inhibiting microbialgrowth and methods for treating and/or preventing microbial infectionsin a subject.

[0061] Those of skill in the art will appreciate that many of thecompounds encompassed by generic formulae (I-III) as well as thecompound species specifically described herein, may exhibit thephenomena of tautomerism, conformational isomerism, geometric isomerismand/or stereo isomerism. As the formula drawings within thespecification and claims can represent only one of the possibletautomeric, conformational isomeric, enantiomeric or geometric isomericforms, it should be understood that the invention encompasses anytautomeric, conformational isomeric, enantiomeric and/or geometricisomeric forms of the compounds having one or more of the utilitiesdescribed herein, as well as mixtures of these various different forms.

4.2.1 Laspartomycin Core Peptide Derivatives

[0062] Laspartomycin core peptide derivatives provide synthetic accessto a wide variety of antimicrobial laspartomycin derivatives that maypossess greater activity against resistant species than previouslydescribed antibiotic agents. The simplicity with which a wide variety ofisolated antimicrobial laspartomycin derivatives can be synthesized fromlaspartomycin core peptide derivatives may establish astructure-activity relationship for the lipophilic group and/or thelinker and linking group. Thus, access to laspartomycin core peptidederivatives may allow for facile investigation of the medicinalchemistry of antimicrobial laspartomycin derivatives.

[0063] Laspartomycin core peptide derivatives include compoundsdescribed by structural Formula (I):

Y ¹—L—X¹—N(R¹)—R  (I)

[0064] or a salt or hydrate thereof, wherein either:

[0065] (i) Y¹—L—X¹ taken together is hydrogen; or

[0066] (ii) Y¹ is a linking group;

[0067] L is a linker;

[0068] X¹ is selected from the group consisting of —CO—, —SO₂—, —CS—,—PO—, —OPO—, —OC(O)—, —NHCO— and —NR¹CO—;

[0069] N is nitrogen;

[0070] R¹ is selected from the group consisting of hydrogen, (C₁-C₁₀ )alkyl optionally substituted with one or more of the same or differentR² groups, (C₁-C₁₀) heteroalkyl optionally substituted with one or moreof the same or different R² groups, (C₅-C₁₀) aryl optionally substitutedwith one or more of the same or different R² groups, (C₅-C₁₅) arylaryloptionally substituted with one or more of the same or different R²groups, (C₅-C₁₅) biaryl optionally substituted with one or more of thesame or different R² groups, five to ten membered heteroaryl optionallysubstituted with one or more of the same or different R² groups,(C₆-C₁₆) arylalkyl optionally substituted with one or more of the sameor different R² groups and six to sixteen membered heteroarylalkyloptionally substituted with one or more of the same or different R²groups;

[0071] each R² is independently selected from the group consisting of—OR³, —SR³, —NR³R³, —CN, —NO₂, —N₃, —C(O)OR³, —C(O)NR³R³, —C(S)NR³R³,—C(NR³)NR³R³, —CHO, R³CO—, —SO₂R³, —SOR³, —PO(OR³)₂, —PO(OR³), —CO₂H,—SO₃H, —PO₃H, halogen and trihalomethyl;

[0072] each R³ is independently selected from the group consisting ofhydrogen, (C₁-C₆) alkyl, (C₅-C₁₀) aryl, 5-10 membered heteroaryl,(C₆-C₁₆) arylalkyl and six to sixteen membered heteroarylalkyl; and

[0073] R is the core cyclic peptide of laspartomycin.

[0074] Those of skill in the art will appreciate that the compounds ofFormula (I) possess the core cyclic peptide of laspartomycin 5 shownbelow as a common structural motif.

[0075] Although the core cyclic peptide R is illustrated as comprised ofcertain amino acids arranged with a particular connectivity, thespecific structure depicted is not intended to be limiting. Thus, itwill be understood that the illustrated structure is intended merely asa convenient method for representing the actual compound and to theextent it may be found at a later date that this structuralrepresentation of the core cyclic peptide of laspartomycin is incorrect,it is not intended to be limiting in any way.

[0076] The moiety covalently bonded to the dashed line of structure 5which represents the core cyclic peptide R in generic formula I isN(R¹). Here, N represents nitrogen that is directly attached to the corecyclic peptide R and R¹ is a nitrogen substituent.

[0077] In a preferred embodiment, R¹ is selected from the groupconsisting of hydrogen, (C₁-C₆) alkyl optionally substituted with one ormore of the same or different R² groups, (C₃-C₇) alkenyl optionallysubstituted with one or more of the same or different R² groups, C₆ aryloptionally substituted with one or more of the same or different R²groups, C₁₂ biaryl optionally substituted with one or more of the sameor different R² groups, (C₆-C₁₀) arylalkyl optionally substituted withone or more of the same or different R² groups and (C₆-C₁₀)heteroarylalkyl optionally substituted with one or more of the same ordifferent R² groups. Preferably, R¹ is selected from the groupconsisting of hydrogen, methyl, allyl, homoallyl, phenyl, substitutedphenyl, benzyl and substituted benzyl. More preferably, R¹ is hydrogen.

[0078] Laspartomycin core peptide derivatives may be H—N(R¹)—R whenY¹—L—X¹ taken together are hydrogen. Preferably, R¹ is hydrogen. Thoseof skill in the art will appreciate that in this situation thelaspartomycin core peptide derivative may be represented by thestructural formula 6 shown below, which is identical to thelaspartomycin core peptide produced by deacylation of laspartomycin withActinoplanes utahensis (NRRL 12052), supra.

[0079] In an alternative embodiment, laspartomycin core peptidederivatives may be described by the formula Y¹—L—X¹—N(R¹)—R. Generally,X¹ may be any kind of chemical functionality that can form a covalentbond with nitrogen known to those of skill in the art. In a exemplaryembodiment, X¹ is selected from the group consisting of —CO—, —SO₂—,—CS—, —PO—, —OPO—, —OC(O)—, —NHCO—, —NR¹CO—. Preferably, X¹ is —CO—or—SO₂—. More preferably, X¹ is —CO—.

[0080] Connected to X¹ in laspartomycin core peptide derivatives of theform Y¹—L—X¹—N(R¹)—R is a linking moiety of the formula Y¹—L, where L isa linker and Y¹ is a linking group. The nature of linker L and linkinggroup Y¹ may vary extensively. The linker L may be hydrophilic orhydrophobic, long or short, rigid, semirigid or flexible.

[0081] A wide variety of linkers L comprised of stable bonds suitablefor spacing linking groups such as Y¹ from the core cyclic peptide areknown in the art, and include by way of example and not limitation,alkyl, heteroalkyl, acyclic heteroatomic bridges, aryl, arylaryl,arylalkyl, heteroaryl, heteroaryl-heteroaryl, substitutedheteroaryl-heteroaryl, heteroarylalkyl, heteroaryl-heteroalkyl and thelike. Thus, linker L may include single, double, triple or aromaticcarbon-carbon bonds, nitrogen-nitrogen bonds, carbon-nitrogen,carbon-oxygen bonds and/or carbon-sulfur bonds, and may therefor includefunctionalities such as carbonyls, ethers, thioethers, carboxamides,sulfonamides, ureas, urethanes, hydrazines, etc.

[0082] Choosing a suitable linker is within the capabilities of thosehaving skill in the art. For example, where a rigid linker is desired, Lmay be a rigid polyunsaturated alkyl or an aryl, biaryl, heteroaryl etc.Where a flexible linker is desired, L may be a flexible peptide such asGly-Gly-Gly or a flexible saturated alkanyl or heteroalkanyl.Hydrophilic linkers may be, for example, polyalcohols or polyethers suchas polyalkyleneglycols. Hydrophobic linkers may be, for example, alkylsor aryls.

[0083] Preferably, linking group Y¹ is capable of mediating formation ofa covalent bond with complementary reactive functionality of alipophilic group to provide an isolated antimicrobial laspartomycinderivative. Accordingly, linking group Y¹ may be any reactive functionalgroup known to those of skill in the art. Y¹ may be for example, aphotochemically activated group, an electrochemically activated group, afree radical donor, a free radical acceptor, a nucleophilic group or anelectrophilic group. However, those of skill in the art will recognizethat a variety of functional groups which are typically unreactive undercertain reaction conditions can be activated to become reactive. Groupsthat can be activated to become reactive include, e.g., alcohols,carboxylic acids and esters, including salts thereof.

[0084] Thus, in a preferred embodiment, Y¹ is selected from the groupconsisting of —NHR¹, —NH₂, —OH, —SH, —PH, halogen, —CHO, —R¹CO, —SO₂H,—PO₂H, —N₃, —CN, CO₂H, —SO₃H, —PO₃H, —PO₂(OR¹)H, —CO₂R¹, —SO₃R¹ and—PO(OR¹)₂. Preferably, Y¹ is selected from the group consisting of—NHR¹, —NH₂, —OH, —SH, —CHO, —CO₂H, R¹CO— and —CO₂R¹. More preferably,Y¹ is selected from the group consisting of —SH, —NH₂, —OH, —CO₂H, and—CO₂R¹.

[0085] Some embodiments of Y¹—L include for example, compounds where Lis —(CH₂)_(n)—, n is an integer between 1 and 8, Y¹ is selected from thegroup consisting of —NH₂, —OH, —CO₂H, and —CO₂R¹ and the correspondinganalogues where any suitable hydrogen is substituted. Other embodimentsof Y¹—L include any amino acid, which may be for example, a D or Lα-amino acid, a β-amino acid or a γ-amino acid. Thus, Y¹—L may be adipeptide, a tripeptide or a tetrapeptide comprised of any combinationof amino acids (preferably α-amino acids). The polarity of the peptidebond in these peptides may be either C→N or N→C.

[0086] In a preferred embodiment of the laspartomycin core peptidederivative, R¹ is hydrogen, Y¹ is selected from the group consistingH₂N—, —OH, —SH, —CO₂H, —CO₂R, X¹ is —CO— and L is selected from thegroup consisting of:

[0087] or a salt or hydrate thereof, wherein:

[0088] n is 0, 1, 2or 3;

[0089] each S¹ is selected from the group consisting of hydrogen,(C₁-C₁₀) alkyl optionally substituted with one or more of the same ordifferent R⁴ groups, (C₁-C₁₀) heteroalkyl optionally substituted withone or more of the same or different R⁴ groups, (C₅-C₁₀) aryl optionallysubstituted with one or more of the same or different R⁴ groups,(C₅-C₁₅) arylaryl optionally substituted with one or more of the same ordifferent R⁴ groups, (C₅-C,₅) biaryl optionally substituted with one ormore of the same or different R⁴ groups, five to ten membered heteroaryloptionally substituted with one or more of the same or different R⁴groups, (C₆-C₁₆) arylalkyl optionally substituted with one or more ofthe same or different R⁴ groups and six to sixteen memberedheteroarylalkyl optionally substituted with one or more of the same ordifferent R⁴ groups;

[0090] each R⁴is independently selected from the group consisting of—OR⁵, —SR⁵, —NR⁵R⁵, —CN, —NO₂, —N₃, —C(O)OR⁵, —C(O)NR⁵R⁵, —C(S)NR⁵R⁵,—C(NR⁵)NR⁵R⁵, —CHO, —R⁵CO, —SO₂R⁵, —SOR⁵, —PO(OR⁵)₂, —PO(OR⁵), —CO₂H,—SO₃H, —PO₃H, halogen and trihalomethyl;

[0091] each R⁵ is independently selected from the group consisting ofhydrogen, (C₁-C₆) alkyl, (C₅-C₁₀) aryl, 5-10 membered heteroaryl,(C₆-C₁₆) arylalkyl and six to sixteen membered heteroarylalkyl; and

[0092] each K is independently selected from the group consisting ofoxygen, nitrogen, sulfur and phosphorus.

[0093] In a preferred embodiment, S¹ is a side chain of a geneticallyencoded α amino acid. Exemplary preferred embodiments of Y¹—L—X¹—NH—Rwhere K is independently selected from the group consisting of oxygen,nitrogen and sulfur include the following compounds:

[0094] Preferably, in the above illustrated embodiments, Y¹ is selectedfrom the group consisting of —SH, —NH₂ or —OH. More preferably Y¹ is—OH.

[0095] In another preferred embodiment of the laspartomycin core peptidederivative, R¹ is hydrogen, Y¹ is H₂N—, X¹ is —CO—, n is as previouslydefined, each S¹ is independently as previously defined and L is L1 aspreviously defined. Preferably, in this embodiment, each S¹ isindependently a side-chain of a genetically encoded α-amnino acid. Morepreferably, each S¹ is independently a side-chain of glycine,asparagine, aspartic acid, glutamine, glutamic acid, tryptophan,phenylalanine, tyrosine, leucine, alanine, isoleucine or valine.Exemplary preferred embodiments of Y¹—L—X¹—N(H)—R where each S¹ isindependently a side-chain of glycine, asparagine, aspartic acid,glutamine, glutamic acid and tryptophan include the following compoundswhere R and Y¹ are as previously defined:

[0096] Preferably, Y¹ is selected from the group consisting of —OH, —SH,—NHR¹ and —NH₂. Most preferably, Y¹ is —NH₂ and the a amino acidsillustrated have the L stereochemistry.

4.2.2 Methods Of Making The Lasartomycin Core Peptide

[0097] The present invention provides methods for making a laspartomycincore peptide that includes culturing the microorganism Streptomycesviridochromogenes, ssp. komabensis (ATCC 29814) in a culture medium toprovide laspartomycin. Isolation of laspartomycin followed by cleavageof a lipophilic fragment provides the laspartomycin core peptide.

[0098] Parent cultures of Streptomyces viridochromogenes, ssp.komabensis (ATCC 29814) especially suitable for biochemical synthesis oflaspartomycin may be selected by conventional methods known to those ofskill in the art. A preferred method for selecting a parent culturewhich provides improved yields of laspartomycin is described in Example1.

[0099] Growing inocula and inoculating culturing medium are also wellknown to those of skill in the art and exemplary methods forStreptomyces viridochromogenes, ssp. komabensis are described in Umezawaet al., U.S. Pat. No. 3,639,582, which is herein incorporated byreference, and Example 2.

[0100] Generally, any culturing medium which supports Streptomycesviridochromogenes, ssp. komabensis growth may be used in the biochemicalsynthesis of laspartomycin and selection of such medium is within thecapability of those of skill in the art. Representative examples ofculturing media which supports Streptomyces viridochromogenes, ssp.komabensis growth may be found in Umezawa et al., U.S. Pat. No.3,639,582 and Examples 3 and 4.

[0101] Preferred media, times, temperatures and pH for culturingStreptomyces viridochromogenes, ssp. komabensis that provide good yieldsof laspartomycin are described in Umezawa et al., U.S. Pat. No.3,639,582 and Examples 3 and 4. It should be noted that the choice ofculturing medium and the quantitative ratio of its constituents directlyaffects the ratio of the different lipopeptides that compriselaspartomycin.

[0102] Generally, laspartomycin may be purified and isolated by anyart-known techniques such as high performance liquid chromatography,counter current extraction, centrifugation, filtration, precipitation,ion exchange chromatography, gel electrophoresis, affinitychromatography and the like. The actual conditions used to purifylaspartomycin will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, etc., and will be apparent to thosehaving skill in the art.

[0103] Preferably, laspartomycin is isolated from the culture medium byextractive procedures. In one preferred embodiment (see e.g., Example5), the fermentation broth containing laspartomycin is mixed withorganic solvent (preferably 1-butanol). While not wishing to be bound bytheory, the anionic form of laspartomycin may form a chelate withdivalent metal ion that is soluble in organic solvent. The organic phasecontaining laspartomycin is then combined with an aqueous acid solutionat a pH less than about 3.0 (most preferably at a pH of about 2.0).While not wishing to be bound by theory, protonation of the anion oflaspartomycin may disrupt the divalent metal chelate form.

[0104] More preferably, (see e.g., Example 6), the fermentation brothcontaining laspartomycin is acidified to a pH of at least about 3.0(more preferably to a pH of about 2.0). The cells and any precipitatemay then be separated by any conventional method known to those of skillin the art and suspended in water. The pH of the aqueous suspension isadjusted to at least about pH 7.0, a divalent metal ion is added and thepH of the aqueous suspension is adjusted to about 8.0 to about 9.0.Preferably the concentration of divalent metal ion in the aqueoussuspension is between about 4 mmol/l and about 10 mmol/l. In oneembodiment, the divalent metal ion is selected from the group consistingof calcium, magnesium and zinc. Most preferably, the divalent metal ionis calcium. The aqueous suspension is then extracted with organicsolvent (preferably, 1-butanol). The organic phase containinglaspartomycin is then combined with an aqueous acid solution at a pHless than about 3.0 (most preferably at a pH of about 2.0).

[0105] Henceforth, in either of the above preferred embodiments,laspartomycin may be partitioned between organic solvent and aqueoussolution by conventional methods known to those of skill in the art.Thus, for example, when the organic solvent solution of laspartomycin istreated with a neutral or basic aqueous solution, laspartomycin may beextracted into aqueous solution. Acidification of the aqueous solutionof laspartomycin enables extraction of laspartomycin into organicsolvent. Preferably, laspartomycin is partitioned between organicsolvent and aqueous solution at least twice. Laspartomycin may beisolated as either the free acid (see e.g. Example 7) or a metal salt(see e.g., Examples 5 and 6) using conventional methods known to thoseof skill in the art.

[0106] Generally, the lipophilic moiety of laspartomycin may be cleavedwith an enzyme to provide the laspartomycin core peptide. It should benoted that addition of an appropriate enzyme to the culture medium mayprovide the laspartomycin core peptide directly, thus obviating the needto isolate laspartomycin. Preferably, however, isolated laspartomycin istreated with an enzyme which may be selected by those of skill in theart. The enzyme may be, for example, a degradative enzyme such as apeptidase, esterase or thiolase, of which numerous examples exist in theart. Preferably, the enzyme is a deacylase.

[0107] In an exemplary embodiment, the cleavage step involves culturinga microorganism that can produce a deacylase in an appropriate culturemedium and contacting laspartomycin with the culture medium containingthe deacylase. Microorganisms that produce deacylases are well known tothose of skill in the art. In a preferred embodiment, the microorganismActinoplanes utahensis (NRRL 12052) provides a deacylase.

[0108] Parent cultures of Actinoplanes utahensis (NRRL 12052) especiallysuitable for cleaving the lipophilic fragment of laspartomycin may beselected by methods known to those of skill in the art. A preferredmethod for selecting a parent culture which provides improved yields oflaspartomycin core peptide is described in Example 8.

[0109] Growing inocula and inoculating culturing medium are also wellknown to those of skill in the art and exemplary methods forActinoplanes utahensis (NRRL 12052) are described in Boeck et al., 1988,J. Antibiot., 41, 1085 and Debono et. al., 1988, J. Antibiotics, 41,1093 which are herein incorporated by reference and Example 8.

[0110] Any culturing medium which supports Actinoplanes utahensis (NRRL12052) growth may be used and selection of such medium is within thecapability of those of skill in the art. Representative examples ofculturing medium which supports Actinoplanes utahensis (NRRL 12052)growth maybe found in Boeck et al., 1988, J. Antibiot., 41, 1085, Debonoet. al., 1988, J. Antibiotics, 41, 1093 and Example 8.

[0111] Preferred media, times, temperatures and pH for culturingActinoplanes utahensis (NRRL 12052) that provide good yields of thedeacylase are described in Boeck et al., 1988, J. Antibiot., 41, 1085,Debono et. al., 1988, J. Antibiotics, 41, 1093 and Example 8.

[0112] In a preferred embodiment, laspartomycin is contacted with aculture medium containing Actinoplanes utahensis (NRRL 12052) for about16 hours at about 29° C. to provide the laspartomycin core peptidehaving the structure:

[0113] It should be noted that contacting laspartomycin with a culturemedium containing Actinoplanes utahensis (NRRL 12052) for about 4 hoursat about 29° C. (see e.g., Example 10) provides material enriched in thelaspartomycin core peptide having the structure:

[0114] While not wishing to be bound by theory, the deacylase producedby Actinoplanes utahensis (NRRL 12052) may be an exopeptidase that firstcleaves the lipophilic fragment of laspartomycin to provide 54. Theexocyclic aspartic acid residue of 54 is then hydrolyzed by extendedtreatment with deacylase or proteases to provide compound 6.

[0115] The laspartomycin core peptide may be purified and isolated byany art-known techniques such as high performance liquid chromatography,counter current extraction, centrifugation, filtration, precipitation,ion exchange chromatography, gel electrophoresis, affinitychromatography and the like. The actual conditions used to purify thelaspartomycin core peptide will depend, in part, on factors such as netcharge, hydrophobicity, hydrophilicity, etc., and will be apparent tothose having skill in the art. Preferably, the laspartomycin corepeptide is isolated by centrifugation and chromatography on reversephase resin (See e.g., Examples 9 and 10).

4.2.3 Methods Of Making Laspartomycin Core Peptide Derivatives

[0116] Laspartomycin core peptide derivatives may be made starting fromlaspartomycin core peptide 6 or laspartomycin core peptide 54.Typically, either 6 or 54 will be produced by deacylation oflaspartomycin provided by culturing Streptomyces viridochromogenes, ssp.komabensis (ATCC 29814). However, it may be possible to synthesizeeither 6 or 54 using methods known in the art for synthesizing cyclicpeptides. For example, linear peptides may be prepared using solutionphase or solid phase peptide synthesis and then cyclized. Preferably,laspartomycin core peptide 6 will be used as a starting material for thesynthesis of laspartomycin core peptide derivatives. Those of skill inthe art will realize that any of the methods presented below can also beused to prepare laspartomycin core peptide derivatives from intermediate54.

[0117] Starting materials useful for preparing laspartomycin corepeptide derivatives from the laspartomycin core peptide 6 andintermediates thereof are either commercially available or may beprepared by conventional synthetic methods. A number of generalsynthetic approaches may be envisioned for converting cyclic peptide 6to laspartomycin core peptide derivatives. These include but are notlimited to the approaches outlined in Schemes I-III.

Scheme 1 Y¹—L—X^(1′)+NH(R¹)—R →Y¹—L—X¹N(R¹)—R

[0118] In Scheme 1, X¹ may be an activated derivative of X¹ such as forexample, —CO—Z, —OCO—Z, —SO₂—Z, —CS—Z, —PO—Z, —OPO—Z, —OC(O)—Z, —NHCO—Zor —NR¹CO—Z where Z is a leaving group such as halogen or an activatedester. Methods for making activated derivatives of X¹ and for reactingthese derivatives with either primary or secondary amines to form theX¹—N covalent bond are known to those of skill in the art and may befound in any compendium of standard synthetic methods (See e.g., March,J., Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4thed., 1992; Larock, R., Comprehensive Organic Transformations, VCH: NewYork, 1999; Bodanzsky, M., Principles of Peptide Synthesis; SpringerVerlag, 1984; Bodanzsky, M., Practice of Peptide Synthesis; SpringerVerlag, 1984). Other synthetic methods based on free radical chemistry,photochemistry or electrochemistry for forming the X¹—N bond will beapparent to those of skill in the art.

[0119] Those of skill in the art will appreciate that protection ofeither Y¹ and/or L may be necessary to make activated derivatives of X¹for formation of the X¹—N bond. In the event that protection of eitherY¹ and/or L is necessary to form the X¹—N linkage, then deprotection ofeither Y¹ and/or L will be necessary to provide the desiredlaspartomycin core peptide derivative. Methods for protection anddeprotection of common organic functionalities are known to those ofskill in the art and may be used as necessary in the synthesis oflaspartomycin core peptide derivatives (see e.g. Greene, T. W.,Protective Groups in Organic Synthesis, 3rd edition, 1999).

4.2.4 Scheme 2 Y¹—L³+L²X¹—N(R¹)R →Y¹—L—X¹N(R¹)—R

[0120] Scheme 2 describes a convergent approach where Y¹—L—X¹N(R¹)—R issynthesized by combining two molecules (Y¹—L³ and L²X¹—N(R¹)R) to formthe laspartomycin core peptide derivative. Here L³ and L² are fragmentswhich, when covalently linked, form the linker L. Such approaches may beparticularly useful when L is an oligomer such as a polyamide orpoylether. Methods for combining oligomeric subunits such as ether oramide monomers, dimers etc. are known to those of skill in the art.Fragments such as Y¹—L³ and L²—X^(1′) (useful in forming the X¹—N bondas described above) are either commercially available or may be made bystandard synthetic methods.

Scheme 3 Y^(X)—L—X¹N(R¹)—R →Y¹—L—X¹N(R¹)—R

[0121] Finally, simple functional group interchange may be used toprepare Y¹—L—X¹N(R¹)—R from Y^(X)—L—X¹N(R¹)—R. Here, Y^(X) is afunctional group that may be converted to Y¹. Many methods for effectingfunctional group interchange are known to those of skill in organicsynthesis (See e.g., March, J., Advanced Organic Chemistry; Reactions,Mechanisms and Structure, 4th ed., 1992; Larock, R., ComprehensiveOrganic Transformations, VCH: New York, 1999).

The Antimicrobial Laspartomycin Derivatives

[0122] The antimicrobial laspartomycin derivatives of the presentinvention offer some significant advantages over traditionalantibiotics. The antimicrobial laspartomycin derivatives are generallyactive against many gram positive bacteria. More importantly, theantimicrobial laspartomycin derivatives of the present invention may beeffective against methicillin resistant bacteria and/or strainsresistant to vancomycin. Thus, the antimicrobial laspartomycinderivatives may inhibit or prevent growth of a number of microbesgenerally resistant to known antibiotics.

[0123] Antimicrobial laspartomycin derivatives include compoundsdescribed by structural Formula (II):

Y ²—(X²—X³)—(L)—X¹)—N(R¹)—R  (II)

[0124] or an pharmaceutically acceptable salt or hydrate thereof,wherein:

[0125] Y² is a lipophilic group;

[0126] X² is a linked group;

[0127] X³ is a linked group; and

[0128] X¹, L, N, R¹ and R are as defined for Formula (II) in Section4.2.1 of this Application.

[0129] Connected to X¹ in isolated antimicrobial laspartomycinderivatives of Formula (II) is a linking moiety of the formula (X²—X³)where L is a linker and X² and X³ are linked groups that attach alipophilic molecule Y² to the linker L. The nature of linker L and thelinked groups X² and X³ may vary extensively. The linker L has beendescribed and defined in Section 4.2.1 of this Application.

[0130] As will be appreciated by those having skill in the art, alinking moiety such as (X²—X³) will typically be at least bifunctional.Thus, they will have at least one functional group or moiety capable offorming a linkage with the linker and at least one functional group ormoiety capable of forming a linkage with a lipophilic group.

[0131] Preferably, linking moiety (X²—X³) taken together is a covalentlinkage. In this preferred embodiment, linking moiety (X²—X³) is anycovalent linkage that may be formed by any method known to those ofskill in the art. Thus, for example, linking moiety (X²—X³) may be anysingle, double or triple bond that can be formed between two carbonatoms, a carbon atom and a heteroatom or two heteroatoms. For example,(X²—X³) include linkages such as —CH₂-CH₂—, —CH═CH—, —C═CH—, —CH═CH—,—C≡C—, —NH -CH₂—, —N═CH—, —CH₂-NH—, —CH═N—, —NH-NH—, —N═N—, —S-S—,—O-O—, —Se-Se—, —S-CH₂—, —CH₂-S—, —O-CH₂—, —CH₂-O—, —Se-CH₂—, —CH₂-Se—,—NH-S—, —P-N—, —N-O— and the corresponding substituted analogs where anysuitable hydrogen is substituted with the same or different substituent.

[0132] Preferably, (X²—X³) taken together are selected from the groupconsisting of —C(O)O—, —O(O)C—, —CONH—, —NHCO—, —CONR¹—, —NR¹CO—,—C(O)S—, —S(O)C—, —OS₂—, —S(O₂)O—, —NHSO₂—, —NR¹SO₂—, —S(O₂)NH—,—S(O₂)NR¹—, —C(S)NH—, —NHC(S)—, —NHP(O)—, —P(O)NH—, OP(O)—, —P(O)O—,SP(O)—, —P(O)S—, —OC(O)NH—, —NHC(O)O—, —OC(O)NR¹—, —NR¹C(O)O—, —OC(O)O—,—NHC(O)NH—, —NHC(O)NR¹ 13 , —NR¹C(O)NH— and —NR¹C(O)NR¹ and thecorresponding substituted analogs where any suitable hydrogen issubstituted with the same or different substituent. In a preferredembodiment, (X²—X³) taken together are selected from the groupconsisting of —C(O)O—, —O(O)C—, CONH—, —NHCO—, —CONR¹—, —NR¹CO—,—C(O)S—, —S(O)C—, —NHSO₂, —NR¹SO₂, —S(O₂)NH—, —S(O₂)NR¹—, C(S)NH—,—NHC(S)—, —OC(O)NH—, —NHC(O)O—, —OC(O)NR¹—, —NR¹C(O)O— and —OC(O)O— andthe corresponding substituted analogs where any suitable hydrogen issubstituted with the same or different substituent. In another preferredembodiment, (X²—X³) taken together are selected from the groupconsisting of —C(O)O—, —O(O)C—, —CONH—, —NHCO—, —CONR¹—, —NR¹CO—,—NHSO₂—, —NR¹SO₂, —S(O₂)NH—, —S(O₂)NR¹—, —OC(O)NH—, —NHC(O)O—, —OC(O)NR—and —NR¹C(O)O— and the corresponding substituted analogs where anysuitable hydrogen is substituted with the same or different substituent.

[0133] Some embodiments of the linking moiety (X²—X³) combined withlinker L include partial structures such as —(X²—X¹)—(CH₂)_(n)—, where nis between 1 and 8, (X²X³) taken together are selected from the groupconsisting of —C(O)O—, —O(O)C—, —CONH—, —NHCO—, —CONR¹—, —NR¹CO—,—NHSO₂—, —NR¹SO₂, —S(O₂)NH—, —S(O₂)NR¹—, —OC(O)NH—, —NHC(O)O—, —OC(O)NR¹and —NR¹C(O)O— and the corresponding analogues where any suitablehydrogen is substituted. Other embodiments of the linking moiety (X²—X³)combined with linker L include representations where X³L taken togetherare derived from any amino acid, which may be for example, a D or Lα-amino acid, a β-amino acid and a γ-amino acid and X², for example is—CO— or —SO₂—. Taken together X³—L also may also be a dipeptide, atripeptide or a tetrapeptide derivative comprised of any combination ofamino acids. The polarity of the peptide bond in these peptides may beeither C→N or N→C.

[0134] Generally, the lipophilic group Y² will be hydrophobic and whensubstituted will be substituted with hydrophobic substituents. Those ofskill in the art will appreciate that the size and/or length of thelipophilic group will depend, in part, on the nature of fragments suchas L, (X²—X³), X¹ and R¹ that comprise the antimicrobial laspartomycinderivatives.

[0135] In a preferred embodiment, the lipophilic group Y² is selectedfrom the group consisting of (C₆-C₂₅) alkyl optionally substituted withone or more of the same or different R² groups, (C₆-C₂₅) heteroalkyloptionally substituted with one or more of the same or different R²groups, (C₈-C₂₅) aryl optionally substituted with one or more of thesame or different R²Agroups, (C₈-C₂₅) arylaryl optionally substitutedwith one or more of the same or different R² groups, (C₈-C₂₅) biaryloptionally substituted with one or more of the same or different R²groups, eight to twenty five membered heteroaryl optionally substitutedwith one or more of the same or different R² groups, (C₈-C₂₅) arylalkyloptionally substituted with one or more of the same or different R²groups and eight to twenty five membered heteroarylalkyl optionallysubstituted with one or more of the same or different R² groups;

[0136] each R² is independently selected from the group consisting of—OR³, —SR³, —NRR³, —CN, —NO₂, —N₃, —C(O)OR³, -C(O)NR³R³, —C(S)NR³R³,—C(NR³)NR³R³, —CHO, —R³CO, —S₂R³, —SOR³, —PO(OR³)₂, —PO(OR³), —CO₂H,—SO₃H, —PO₃H, halogen and trihalomethyl;

[0137] each R³ is independently selected from the group consisting ofhydrogen, (C₁-C₆) alky, (C₅-C₁₀) aryl, 5-10 membered heteroaryl,(C₆-C₁₆) arylalkyl and 6-16 membered heteroarylalkyl.

[0138] In a more preferred embodiment, the lipophilic group Y² isselected from the group consisting of (C₈-C₂₀) alkyl optionallysubstituted with one or more of the same or different R² groups,(C₈-C₂₀) heteroalkyl optionally substituted with one or more of the sameor different R² groups, (C₈-C₂₀) aryl optionally substituted with one ormore of the same or different R² groups, (C₈-C₂₀) arylaryl optionallysubstituted with one or more of the same or different R² groups,(C₈-C₂₀) biaryl optionally substituted with one or more of the same ordifferent R² groups, eight to twenty membered heteroaryl optionallysubstituted with one or more of the same or different R² groups,(C₈-C₂₀)arylalkyl optionally substituted with one or more of the same ordifferent R² groups and eight to twenty membered heteroarylalkyloptionally substituted with one or more of the same or different R²groups where R² is as defined above.

[0139] In one preferred embodiment, the lipophilic group Y² is selectedfrom the group consisting of (C₈-C₂₀) alkyl optionally substituted withone or more of the same or different R² groups, (C₈-C₂₀) heteroalkyloptionally substituted with one or more of the same or different R²groups, (C₈-C₂₀) aryl optionally substituted with one or more of thesame or different R² groups, (C₈-C₂₀) arylaryl optionally substitutedwith one or more of the same or different R² groups, (C₈-C₂₀) biaryloptionally substituted with one or more of the same or different R²groups, ten to twenty membered heteroaryl optionally substituted withone or more of the same or different R² groups, (C₈-C₂₀) arylalkyloptionally substituted with one or more of the same or different R²groups and ten to twenty membered heteroarylalkyl optionally substitutedwith one or more of the same or different R² groups. In anotherpreferable embodiment, the lipophilic group Y² is selected from thegroup consisting of (C₈-C₂₀) alkyl optionally substituted with one ormore of the same or different R² groups. In yet another preferableembodiment, the lipophilic group Y² is selected from the groupconsisting of (C₁₀-C₁₆) alkyl optionally substituted with one or more ofthe same or different R² groups.

[0140] In an exemplary embodiment of the isolated antimicrobiallaspartomycin derivative of Formula (II), X¹ is —CO— or —SO₂—, (X²—X³)taken together are selected from the group consisting of —C(O)O—,—O(O)C—, —CONH—, —NHCO—, —C(O)S—, —S(O)C—, —OSO₂—, —S(O₂)O—, —NHS₂—,—S(O₂)NH—, —C(S)NH—, —NHC(S)—, —NHP(O)—, —P(O)NH—, OP(O)—, —P(O)O—,—SP(O)—, —P(O)S—, —OC(O)NH—, —NHC(O)O—, —OC(O)NR¹—, —NR¹C(O)O—,—OC(O)O—, —NHC(O)NH—, —NHC(O)NR¹— and —NR¹C(O)O—, R¹ is hydrogen and Lis selected from the group consisting of L1, L2, L3 and L4 where L1, L2,L3 and L4 are as defined in Section 4.2.1 of this Application

[0141] In a preferred embodiment, S¹ is a side chain of a geneticallyencoded α amino acid. Exemplary preferred embodiments ofY²—(X²—X³)—L—X¹—N(R¹)—R where K is independently selected from the groupconsisting of oxygen, nitrogen and sulfur include the followingcompounds where Y², X², X³ and R are as previously defined:

[0142] Preferably, in the these embodiments, X³ is selected from thegroup consisting of —S—, —O— or —NH—and X² is selected from the groupconsisting of —CO—, —SO₂—, —OC(O)—, —NHC(O)— and —NR¹C(O)—. In analternative embodiment, X² is selected from the group consisting of —S—,—O— or —NH— and X³ is selected from the group consisting of —CO—, —SO₂—,—OC(O)—, —NHC(O)— and —NR¹C(O)—.

[0143] In another preferred embodiment of the antimicrobiallaspartomycin derivatives, X¹ is —CO— or —SO₂—, (X²—X³) taken togetherare selected from the group consisting of —CONH—, —S(₂)NH—, —C(S)NH—,—P(O)NH—, —OC(O)NH—, —OC(O)NR¹—, —NHC(O)NH—, and —NHC(O)NR¹, R¹ ishydrogen, n is as defined in Section 4.2.1 of this Application and L isL1 as defined in Section 4.2.1 of this Application. Preferably, in thisembodiment, each S¹ is independently a side-chain of a geneticallyencoded α-amino acid. More preferably, each S¹ is independently aside-chain of glycine, asparagine, aspartic acid, glutamine, glutamicacid, tryptophan, phenylalanine, tyrosine, leucine, alanine, isoleucineor valine. Exemplary preferred embodiments of Y²—(X²—X³)—L—X¹—N(R¹)—Rwhere each S¹ is independently a side-chain of glycine, asparagine,aspartic acid, glutamine, glutamic acid or tryptophan include thefollowing compounds where Y², (X²—X³) taken together and R are aspreviously defined:

[0144] Preferably, in the these embodiments X³ is selected from thegroup consisting of —S—, —O— or —NH— and X² is selected from the groupconsisting of —CO—, —SO₂—, —OC(O)—, —NHC(O)— and —NR¹C(O)—. Inalternative embodiment, X² is selected from the group consisting of —S—,—O— or —NH— and X³ is selected from the group consisting of —CO—, —SO₂—,—OC(O)—, —NHC(O)— and —NR¹C(O)—. Preferably, in the above depictedembodiments the illustrated a amino acids have the L stereochemistry.

[0145] In a preferred embodiment X²—X³ taken together are —CONH— or—SO₂NH—. Most preferably, X²—X³ taken together are —CONH—. Particularlypreferred embodiments of Y² include tetradecan-1-yl, nonan-1-yl,decan-1-yl and 12-methyl-tridecan-1-yl.

[0146] Exemplary preferred isolated antimicrobial laspartomycinderivatives according to structural formula II include:

[0147] Preferably, in the above depicted embodiments, the polyamidelinkers depicted have the L stereochemistry at the a carbon of theillustrated amino acids.

4.2.5 Methods Of Making Antimicrobial Laspartomycin Derivatives

[0148] Antimicrobial laspartomycin derivatives may be synthesized fromlaspartomycin core peptide 6, laspartomycin core peptide 54 andlaspartomycin core peptide derivatives of Formula (I). Laspartomycincore peptide derivatives of Formula (1) may be synthesized by theapproaches outlined in Section 4.2.2 of this Application. Those of skillin the art will appreciate that other starting materials may be used inthe synthesis of antimicrobial laspartomycin derivatives.

[0149] A number of general synthetic approaches may be envisioned forconverting laspartomycin core peptide 6, laspartomycin core peptide 54and laspartomycin core peptide derivatives of Formula I to antimicrobiallaspartomycin derivatives. These include but are not limited to theapproaches outlined in Schemes 4 and 5.

Scheme 4 Y²—X²—X³—L—X^(1′)+HN(R¹)—R →Y²—X²—X³—L—X¹N(R¹)—R

[0150] In Scheme 4 a lipophilic fragment Y² and a linker L, attached vialinked groups X² and X³ are covalently linked to X^(1′)which may be anactivated derivative of X¹ such as for example, —CO—Z, —OCO—Z,—SO₂—Z—CS—Z, —PO—Z, —OPO—Z, —OC(O)—Z, —NHCO—Z or —NR¹CO—Z where Z is aleaving group such as halogen or an activated ester. Methods for makingactivated derivatives of X¹ and for reacting these derivatives witheither primary or secondary amines to form the X¹—N covalent bond areknown to those of skill in the art and may be found in any compendium ofstandard synthetic methods (See e.g., March, J., Advanced OrganicChemistry; Reactions, Mechanisms and Structure, 4th ed., 1992; Larock,R., Comprehensive Organic Transformations, VCH: New York, 1999;Bodanzsky, M., Principles of Peptide Synthesis; Springer Verlag, 1984;Bodanzsky, M., Practice of Peptide Synthesis; Springer Verlag, 1984).Other synthetic methods based on free radical chemistry, photochemistryor electrochemistry for forming the X¹—N bond will be apparent to thoseof skill in the art. Formation of the X¹—N covalent bond provides theantimicrobial laspartomycin derivative. Methods for making (X²—X³)linkages such as esters, amides phosphoramidites, sulfonamides,carbamates, ureas etc. are also conventional and known to those of skillin the art (See e.g., March, J., Advanced Organic Chemistry; Reactions,Mechanisms and Structure, 4th ed., 1992; Larock, R., ComprehensiveOrganic Transformations; VCH: New York, 1999; Bodanzsky, M., Principlesof Peptide Synthesis; Springer Verlag, 1984; Bodanzsky, M., Practice ofPeptide Synthesis; Springer Verlag, 1984)

Scheme 5 Y²—X^(2′)+Y¹—L—X¹N(R¹)—R →Y²—X²—X³—L—X¹N(R¹)—R

[0151] Scheme 5 describes a convergent approach where Y²—X^(2′)(X^(2′)isa derivative of the linked group X²) and Y¹—L—X¹N(R¹)—R are combined toform the (X²—X³) linkage thus providing the antimicrobial laspartomycinderivative. Methods for forming the (X²—X³) linkage are described above.Fragments such as Y²—X^(2′)are either commercially available or may bemade by standard synthetic methods. Y¹—L—X¹N(R¹)—R may be made asdescribed in Section 4.2.2 of this application.

[0152] Those of skill in the art will appreciate that protection ofeither Y² and/or L may be necessary to form (X²—X³) linkage. In theevent that protection of either Y² and/or L is necessary to form the(X²—X³) linkage, then deprotection of either Y² and/or L will benecessary to provide the antimicrobial laspartomycin derivative. Methodsfor protection and deprotection of common organic functionalities areknown to those of skill in the art and may be used as necessary in thesynthesis of antimicrobial laspartomycin derivatives (see e.g. Greene,T. W., Protective Groups in Organic Synthesis, 3rd edition, 1999).

4.2.6 Methods Of Inhibiting Microbial Growth

[0153] Generally, active isolated antimicrobial laspartomycinderivatives of the invention are identified using in vitro screeningassay. Indeed, in many instances the isolated antimicrobiallaspartomycin derivatives of the invention will be used in vitro aspreservatives, topical antimicrobial treatments, etc. Additionally,despite certain apparent limitations of in vitro susceptibility tests,clinical data indicate that a good correlation exists between minimalinhibitory concentration (MIC) test results and in vivo efficacy ofantibiotic compounds (Murray, 1994, Antimicrobial SusceptibilityTesting, Poupard et al, eds., Plenum Press, NY; Knudsen et al., 1995,Antimicrob. Agents Chemother. 39 (6):1253-1258). Thus, isolatedantimicrobial laspartomycin derivatives useful for treating infectionsand diseases related thereto are also conveniently identified bydemonstrated in vitro antimicrobial activity against specified microbialtargets.

[0154] Generally, the in vitro antimicrobial activity of antimicrobialagents is tested using standard NCCLS bacterial inhibition assays, orMIC tests (see, National Committee on Clinical Laboratory Standards“Performance Standards for Antimicrobial Susceptibility Testing,” NCCLSDocument M100-S5 Vol. 14, No. 16, December 1994; “Methods for dilutionantimicrobial susceptibility test for bacteria that growaerobically-Third Edition,” Approved Standard M7-A3, National Committeefor Clinical Standards, Villanova, Pa.).

[0155] Alternatively, the antimicrobial laspartomycin derivatives of theinvention may be assessed for antimicrobial activity using in vivomodels. Again, such models are well-known in the art.

[0156] It will be appreciated that other assays, that are well known inthe art or which will become apparent to those having skill in the artupon review of this disclosure, may also be used to identify activeisolated antimicrobial laspartomycin derivatives of the invention. Suchassays include, for example, the assay described in Lehrer et al., 1988,J. Immunol. Methods 108:153 and Steinberg and Lehrer, “Designer Assaysfor Antimicrobial Peptides: Disputing the ‘One Size Fits All’ Theory,”In: Antibacterial Peptide Protocols, Shafer, Ed., Humana Press, N.J.

[0157] Generally, isolated antimicrobial laspartomycin derivatives ofthe invention will exhibit MICs of less than about 64 μg/mL, usuallyless than about 32 μg/mL, preferably less than about 16 μg/mL and mostpreferably less than about 4 μg/mL. The antimicrobial laspartomycinderivatives of the invention may also exhibit antifungal activity,having MICs of about 50 μg/mL or less against a variety of fungi instandard in vitro assays.

[0158] Of course, compounds having MICs on the low end of these ranges,or even lower, are preferred. Most preferred for use in treating orpreventing systemic infections are antimicrobial laspartomycinderivatives that exhibit significant antimicrobial activity (i.e., lessthan 4 μg/mL), good water-solubility (at approx. neutral pH) and lowtoxicity. Toxicity is less of a concern for topical administration, asis water solubility.

4.2.7 Other Methods And Pharmaceutical Compositions

[0159] The antimicrobial laspartomycin derivatives of the invention canbe used in a wide variety of applications to inhibit the growth ofmicroorganisms or kill microorganisms. For example, the antimicrobiallaspartomycin derivatives maybe used as disinfectants or aspreservatives for materials such as foodstuffs, cosmetics, medicamentsand other nutrient containing materials. The antimicrobial laspartomycinderivatives can also be used to treat or prevent diseases related tomicrobial infection in subjects such as plants and animals.

[0160] For use as a disinfectant or preservative, the antimicrobiallaspartomycin derivatives can be added to the desired material singly,as mixtures of antimicrobial laspartomycin derivatives, or incombination with other antifungal and/or antimicrobial agents. Theantimicrobial laspartomycin derivatives may be supplied as the compoundper se or may be in admixture with a variety of carriers, diluents orexcipients, which are well known in the art.

[0161] When used to treat or prevent microbial infections or diseasesrelated thereto the antimicrobial laspartomycin derivatives of theinvention can be administered or applied singly, as mixtures of two ormore antimicrobial laspartomycin derivatives, in combination with otherantifungal, antibiotic or antimicrobial agents or in combination withother pharmaceutically active agents. The antimicrobial laspartomycinderivatives can be administered or applied per se or as pharmaceuticalcompositions. The specific pharmaceutical formulation will depend uponthe desired mode of administration, and will be apparent to those havingskill in the art. Numerous compositions for the topical or systemicadministration of antibiotics are described in the literature. Any ofthese compositions may be formulated with the antimicrobiallaspartomycin derivatives of the invention.

[0162] Pharmaceutical compositions comprising the antimicrobiallaspartomycin derivatives of the invention may be manufactured by meansof conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical compositions may be formulated in conventionalmanner using one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the activeantimicrobial laspartomycin derivatives into preparations which can beused pharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

[0163] For topical administration the antimicrobial laspartomycinderivatives of the invention may be formulated as solutions, gels,ointments, creams, suspensions, etc. as are well-known in the art.

[0164] Systemic formulations include those designed for administrationby injection, e.g. subcutaneous, intravenous, intramuscular, intrathecalor intraperitoneal injection, as well as those designed for transdermal,transmucosal, oral or pulmonary administration.

[0165] For injection, the antimicrobial laspartomycin derivatives of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents.

[0166] Alternatively, the antimicrobial laspartomycin derivatives may bein powder form for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

[0167] For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0168] For oral administration, the antimicrobial laspartomycinderivatives can be readily formulated by combining them withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, such aslactose, sucrose, mannitol and sorbitol; cellulose preparations such asmaize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. If desired, solid dosageforms may be sugar-coated or enteric-coated using standard techniques.

[0169] For oral liquid preparations such as, for example, suspensions,elixirs and solutions, suitable carriers, excipients or diluents includewater, glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like may be added.

[0170] For buccal administration, the compositions may take the form oftablets, lozenges, etc. formulated in conventional manner.

[0171] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0172] The antimicrobial laspartomycin derivatives may also beformulated in rectal or vaginal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

[0173] In addition to the formulations described previously, theantimicrobial laspartomycin derivatives may also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compounds may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

[0174] Alternatively, other pharmaceutical delivery systems may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles that may be used to deliver the antimicrobial laspartomycinderivatives of the invention. Certain organic solvents such asdimethylsulfoxide also may be employed, although usually at the cost ofgreater toxicity. Additionally, the antimicrobial laspartomycinderivatives may be delivered using a sustained-release system, such assemipermeable matrices of solid polymers containing the therapeuticagent. Various sustained-release materials have been established and arewell known by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds for a fewweeks up to over 100 days.

[0175] As certain of the carboxylic acids of the antimicrobiallaspartomycin derivatives of the invention are acidic, or the lipophilicgroup or linker may include acidic or basic substituents, theantimicrobial laspartomycin derivatives may be included in any of theabove-described formulations as the free acids, the free bases or aspharmaceutically acceptable salts. Pharmaceutically acceptable salts arethose salts which retain substantially the antimicrobial activity of thefree acids or bases and which are prepared by reaction with bases oracids, respectively. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseor acid forms.

[0176] The antimicrobial laspartomycin derivatives of the invention, orcompositions thereof, will generally be used in an amount effective toachieve the intended purpose. Of course, it is to be understood that theamount used will depend on the particular application.

[0177] For example, for use as a disinfectant or preservative, anantimicrobially effective amount of a antimicrobial laspartomycinderivative, or composition thereof, is applied or added to the materialto be disinfected or preserved. By antimicrobial effective amount ismeant an amount of antimicrobial laspartomycin derivative or compositionthat inhibits the growth of, or is lethal to, a target microbe. Whilethe actual amount will depend on a particular target microbe andapplication, for use as a disinfectant or preservative the antimicrobiallaspartomycin derivatives, or compositions thereof, are usually added orapplied to the material to be disinfected or preserved in relatively lowamounts. Typically, the antimicrobial laspartomycin derivativescomprises less than about 5% by weight of the disinfectant solution ormaterial to be preserved, preferably less than about 1% by weight andmore preferably less than about 0.1% by weight. An ordinarily skilledartisan will be able to determine antimicrobially effective amounts ofparticular antimicrobial laspartomycin derivatives for particularapplications without undue experimentation using, for example, the invitro assays provided in the examples.

[0178] For use to treat or prevent microbial infections, theantimicrobial laspartomycin derivatives of the invention, orcompositions thereof, are administered or applied in a therapeuticallyeffective amount. By therapeutically effective amount is meant an amounteffective to ameliorate the symptoms of, or ameliorate, treat or preventmicrobial infections. Determination of a therapeutically effectiveamount is well within the capabilities of those skilled in the art,especially in light of the detailed disclosure provided herein.

[0179] As in the case of disinfectants and preservatives atherapeutically effective dose, for topical administration to treat orprevent microbial, yeast, fungal or other infection, can be determinedusing, for example, the in vitro assays provided in the examples. Thetreatment may be applied while the infection is visible, or even when itis not visible. An ordinarily skilled artisan will be able to determinetherapeutically effective amounts to treat topical infections withoutundue experimentation.

[0180] For systemic administration, a therapeutically effective dose canbe estimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating antimicrobiallaspartomycin derivative concentration range that includes the IC₅₀ asdetermined in cell culture (i.e., the concentration of test compoundthat is lethal to 50% of a cell culture), the MIC as determined in cellculture (i.e., the minimal inhibitory concentration for growth) or theIC₁₀₀ as determined in cell culture (i.e., the concentration ofantimicrobial laspartomycin derivative that is lethal to 100% of a cellculture). Such information can be used to more accurately determineuseful doses in humans.

[0181] Initial dosages can also be estimated from in vivo data, e.g.,animal models, using techniques that are well known in the art. Onehaving ordinary skill in the art can readily optimize administration tohumans based on animal data.

[0182] Alternatively, initial dosages can be determined from the dosagesadministered of known antimicrobial agents (e.g., laspartomycin) bycomparing the IC₅₀, MIC and/or I₁₀₀ of the specific antimicrobiallaspartomycin derivatives with that of a known antimicrobial agent, andadjusting the initial dosages accordingly. The optimal dosage may beobtained from these initial values by routine optimization.

[0183] Dosage amount and interval may be adjusted individually toprovide plasma levels of the active antimicrobial laspartomycinderivatives which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to5 mg/kg/day, preferably from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective serum levels may be achieved by administering a single dailydose or multiple doses each day.

[0184] In cases of local administration or selective uptake, theeffective local concentration of antimicrobial laspartomycin derivativemay not be related to plasma concentration. One having skill in the artwill be able to optimize therapeutically effective local dosages withoutundue experimentation.

[0185] The amount of antimicrobial laspartomycin derivative administeredwill, of course, be dependent on, among other factors, the subject beingtreated, the subject's weight, the severity of the affliction, themanner of administration and the judgment of the prescribing physician.

[0186] The antimicrobial therapy may be repeated intermittently whileinfections are detectable, or even when they are not detectable. Thetherapy may be provided alone or in combination with other drugs, suchas for example other antibiotics or antimicrobials, or otherantimicrobial laspartomycin derivatives of the invention.

[0187] Preferably, a therapeutically effective dose of the antimicrobiallaspartomycin derivatives described herein will provide therapeuticbenefit without causing substantial toxicity. Toxicity of theantimicrobial laspartomycin derivatives can be determined using standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index.Antimicrobial laspartomycin derivatives which exhibit high therapeuticindices are preferred. The data obtained from these cell culture assaysand animal studies can be used in formulating a dosage range that is nottoxic for use in subjects. The dosage of the antimicrobial laspartomycinderivatives described herein lies preferably within a range ofcirculating concentrations that include the effective dose with littleor no toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition (See, e.g. Finglet al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p.1).

5. EXAMPLES

[0188] The invention having been described, the following examples arepresented to illustrate, rather than to limit, the scope of theinvention. The examples illustrate various embodiments and features ofthe present invention.

5.1 Example 1: Selection Of Parent Culture

[0189] The parent culture used for biochemical synthesis oflaspartomycin is Streptomyces viridochromogenes ssp. komabensis,(ATCC-29814, BSP-M728) which was selected as follows. A cell suspensionof Streptomyces viridochromogenes ssp. komabensis, (ATCC-29814) wasdiluted so that plating on a nutrient medium gave well separated singlecolonies after incubation at about 28° C. A few colonies were isolatedand tested by fermentation for improvement in laspartomycin yield on thebasis of morphological observations (colony size, surface structure,edge profile, etc.) which are within the capabilities of those of skillin the art. The colony BSP-M728/1, provided higher and more reproducibleyields and yielded superior correlation with mycelial densityin thefermentation mash. Thus, for at least these reasons, Streptomycesviridochromogenes ssp. komabensis,BSP-M728/1) was selected forbiochemical synthesis of laspartomycin.

5.2 Example 2: Medium Inoculation

[0190] Ideally, the biochemical synthesis of laspartomycin is performedby inoculating a medium composed of about 3.0% trypticase soy broth,about 1.0% corn dextrin and 0.1% CaCO₃ in tap water with spore andmycelial scrapings from a slant of Streptomyces viridochromogenes ssp.komabensis, (BSP-M728/1). Incubation of about 50 mL of the inoculatedmedium at 28° C. on a rotary shaker at about 200 revolutions per minute(“RPM”) for about 48 hours provides a substantial and uniform vegetativegrowth. The growth may then be used to inoculate various fermentationmedia (See, e.g., Example 3). Preferably, the growth comprises aconcentration range of between about 2.0% to about 3.0% of thefermentation medium when used to inoculate fermentation medium.

5.3 Example 3: Shaker Flask Fermentation

[0191] The inoculum produced in Example 2 may be used to seed a numberof fermentation media such as: (1) a medium containing about 2.0%dextrose, about 0.5% beef extract, about 0.5% Bacto-peptone, about 0.5%NaCl and about 0.35% CaCO₃ in water; (2) a medium containing about 0.5%dextrose, about 1.5% dextrin, about 1.0% molasses, about 1.0%Bacto-peptone and about 0.1% CaCO₃ dissolved in water; and (3) a mediumcontaining about 0.5% dextrose, about 1.5% glycerol, about 0.75%Soytone, about 0.2% NaCl and about 0.1% CaCO₃ in water. In typicalshaker flask fermentations, about 50 mL of the above media are seededwith the inoculum of Example 2 and are incubated at a temperature ofabout 28° C. on a rotary shaker at between about 160 and about 180 RPMfor a period of between about 4 and about 7 days.

5.4 Example 4: Biochemical Synthesis Of Laspartomycin

[0192] Biochemical synthesis of laspartomycin is preferably performed ina culture medium containing about 0.5% dextrose, about 1.5% corndextrin, about 0.75% Soytone, 0.3% NaCl, about 0.1% MgSO_(4,) 7H₂O andabout 0.1% CaCO₃ in water. The unadjusted pH of this medium is generallybetween about 7.2 and about 7.3. The inoculated medium is incubated at atemperature of between about 24° C. to about 34° C. (preferably betweenabout 27° C. to about 29° C., most preferably about 28° C.) on a rotaryshaker at between about 140 and about 200 RPM (preferably between about160 and about 180 RPM) for a period of between about 4 and about 7 days(preferably, between about 5 and about 6 days) until significant amountsof laspartomycin are synthesized. Harvest pH readings of the medium arebetween about 8.0 and about 8.6. The yield for laspartomycin is about600 mg/liter of fermentation medium, while the yield of the C-15laspartomycin derivative is about 400 mg/liter of fermentation medium.The medium formulation and the quantitative ratio of its members has adirect effect on the ratio of the individual lipopeptide components oflaspartomycin.

5.5 Example 5: Separation Of Lasartomycin From Fermentation Broth

[0193] About 1.85 liters of fermentation broth produced by the method ofExample 4 at pH of about 8.5 was mixed with an equal volume of 1-butanoland the phases allowed to separate. The dark brown aqueous phase wasdiscarded and the slightly colored 1-butanol phase containinglaspartomycin was combined with an equal amount of distilled water,stirred and the pH of the mixture was adjusted to about 2.0 with 1 NHCl.The phases were separated and the 1-butanol phase was washed with ¼ itsvolume of water, mixed with an equal volume of water and the pH of themixture was adjusted to about 7.0. The phases again were separated andthe pH of the aqueous phase containing laspartomycin was adjusted toabout 2.0 and laspartomycin was extracted into 1-butanol and then backinto the aqueous phase at a pH of about 7.0. The aqueous phase containedlaspartomycin as the partial sodium salt. The solution was evaporatedunder vacuum to remove residual 1-butanol and then lyophilized toprovide about 561 mg of the sodium salt of laspartomycin as a whitepowder.

5.6 Example 6: Separation Of Laspartomycin From Fermentation Broth

[0194] About 1.8 liters of fermentation broth produced by the method ofExample 4 was adjusted to about pH 2.0 and allowed to stand at about 4°C. for three hours. The cells and any precipitate were separated bycentrifugation and suspended in about 500 mL of water. The pH of thesuspension was adjusted to about 7.0 with 1 N NaOH and the resultingmixture was stirred at room temperature for approximately one hour.Calcium chloride (about 500 mg) was added to the suspension and the pHof the mixture was adjusted to between about 8.6 and about 9.0 with 1.0N NaOH. Laspartomycin was extracted from the aqueous suspension by twosequential washings with about 500 mL and then about 100 mL of1-butanol. While not wishing to be bound by theory, laspartomycin mayform a chelate with the added calcium ion. The combined butanol extractswere mixed with an equal volume of distilled water, adjusted to about pH2.0 with 1 N HCl and rinsed twice with 200 mL of distilled watermaintained at about pH 2.0. While not wishing to be bound by theory, thelaspartomycin calcium chelate may be disrupted by acidic solutions andcalcium ion may be removed by washing with acidic water. The 1-butanolphase containing the antibiotic was separated, mixed with an equalvolume of distilled water and the mixture adjusted to about pH 7.0 with1 N NaOH to provide laspartomycin in the aqueous phase. The aqueousphase was separated and laspartomycin was then extracted into 1-butanolat about pH 3.0 and then into an aqueous phase at about pH 7.0. Theclear almost colorless aqueous phase was evaporated under vacuum toremove residual 1-butanol and freeze-dried to obtain 668 mg of thesodium salt of laspartomycin as a white powder.

[0195] HPLC of the salt indicated that about 80% of the salt was theC-15 component of laspartomycin. High resolution FAB-mass spectroscopy:calculated for C₅₇H₉₀N₁₂O₁₉+Na (M+Na)^(+,) 1269.6343, found, 1269.6289which corresponds to a molecular formula C₅₇H₉₀N_(l2)O₁₉ for the C-15component of laspartomycin.

[0196] Laspartomycin was hydrolyzed with 6N HCl at 120° C. for 16 hours.Amino acid analysis provided the following amino acids in the indicatedmolar ratios: aspartic acid (3 moles), glycine (3 moles), pipecolic acid(1 mole), allo-threonine (1 mole), isoleucine (1 mole), diaminopropionicacid (1 mole) and proline (1 mole).

5.7 Example 7: Prearation Of The Acid Form Of Laspartomycin

[0197] The acid form of laspartomycin was prepared by dissolving about100 mg of the sodium salt prepared as described in Example 6 into about10 mL of water and adjusting the pH of the solution to about 2.0 with0.1 N HCl. The aqueous solution was extracted with about 10 mL of1-butanol. The organic extract was then washed with about 5 mL of water,mixed with about another 20 mL of water, evaporated under vacuum toobtain an aqueous solution of laspartomycin as the carboxylic acid andfreeze-dried to obtain about 77 mg of white powder. FAB-MS m/z: 1248(M+H)^(+,) 1270(M+Na)⁺, and 1286 (M+K)⁺which corresponds to a molecularformula of C₅₇H₉₀N₁₂O₁₉ for the C-15 component of laspartomycin.Elemental analysis: found: C, 52.13; H, 7.58; N, 11.83; O, 28.34.

5.8 Example 8: Selection Of Deacylase Microorganism And BiochemicalSynthesis Of Deacylase

[0198]Actinoplanes utahensis NRRL 12052 was cultured under submergedaerobic fermentation conditions to provide the deacylase. Becausesingle-colony isolates of the culture were heterogeneous for bothmorphology and enzyme production capability, selections were made torecover a stable, high-producing variant. Initially, multiplefermentations were carried out using inocula prepared from strain 12052.Vegetative growth yielding the highest deacylating activity was platedon a differential agar, such as CM agar, which contains 0.5% corn steepliquor, 0.5% Bacto peptone, 1.0% soluble starch, 0.05% NaCl, 0.05%CaCl₂-2H₂O and 2.0% Bacto agar. Colonies were then selected for furtherevaluation. Generally, small colonies were better enzyme producers thanthe large colony types. Isolate No. 18 was the highest deacylaseproducer selected and was routinely used for the production of thedeacylase enzyme.

[0199] The high-producing, natural variant was used in a knownfermentation protocol (Boeck et al., 1988, J. Antibiot., 41, 1085). Amycelial suspension of the high producing NRRL 12052 variant was grownfrom a stock culture (preserved in 20% glycerol at −70° C.) in about 10mL of a medium, which contained about 2.0% sucrose, about 2.0%pre-cooked oatmeal, about 0.5% distiller's grain, about 0.25% yeast,about 0.1% K₂HPO_(4,)0.05% KCl, about 0.05% MgSO₄-7H₂O and about 0.0002%FeSO₄-7H₂O in deionized water at about 30° C. for about 72 hrs on arotary shaker orbiting at about 250 RPM. The mycelial suspension wastransferred to about 50 mL of PM3 medium, which contained about 2%sucrose, about 1.0% peanut meal, about 0.12% K₂HPO₄, about 0.05% KH₂PO₄and about MgSO₄-7H₂O in tap water and incubated at a temperature ofabout 30° C. for a period of about 60 to about 90 hrs.

5.9 Example 9: Synthesis Of Compound 6

[0200] Two hundred fifty-seven milligrams of laspartomycin in about 12mL of 0.5 M phosphate buffer of about pH 7.2 was added to about 120 mLof deacylase fermentation broth prepared as in Example 8 and incubatedfor about 16 hours at about 29° C. at about 180 rpm. The broth wascentrifuged, the centritugate decanted and solids were extracted withabout 40 mL of distilled water. The pooled centrifugates were thenapplied to a 2.5×5.0 cm styrene-divinylbenzene resin column (ENVI™-ChromP) and the product was eluted with a 10% and 11% acetonitrile-pH 7.2phosphate mixture. Pooled fractions were concentrated and the pH wasadjusted to about 4.65 by addition of ammonium acetate-acetic acidbuffer. The fractions were then applied to a 2.5×5.0 cm resin column(ENVI™-Chrom P). The desired material was eluted with a 12.5%acetonitrile-pH 4.65 acetate mixture. The pH of the pooled fractions wasadjusted to about 7.8, followed by concentration and freeze-dried toprovide about 74 mg of 6 as an off-white solid which was about 97% purewhen analyzed by High Pressure Liquid Chromatography (“HPLC”) at 215 nm.FAB-MS m/z 910 (HR-FAB-MS of 6: found 910.4251(M+H)⁺, calc. 910.4270 forC₃₈H₅₉N₁₁O₁₅+H). Also obtained was about 14 mg of an isomer of 6 as anoff white solid. FAB-MS: m/z 910(M+H)⁺.

5.10 Example 10: Synthesis of Compound 6 And Compound 54

[0201]

[0202] About 2.5g of laspartomycin was treated with the deacylase brothunder conditions similar to those described in Example 9 except whereexplicitly noted. About 1.0 g of laspartomycin was treated withdeacylase at about 2.0 mg/mL for about 3.7 hrs to produce a sampleenriched in 54. About 1.5 g of laspartomycin was treated with deacylaseat about 5.0 mg/mL for about 20 hours. The fermentation broths werepooled and then processed as described in Example 9 to provide about 100mg of 54, about 600 mg of 6, and an estimated 150 mg of the isomer of 6.FAB-MS of 54: m/z 1026(M+H)^(+,) 1048(M+Na)⁺.

5.11 Example 11: Synthesis Of Pentadecanoyl-L-Aspartic-Acid-4-O-BenzylEster

[0203] Equimolar amounts of pentadecanoic acid,dicyclohexylcarbodiimide, and 1-hydroxybenzotriazole in tetrahydrofuranwas stirred overnight and the reaction mixture was filtered andevaporated to give a crystalline solid. The solid was then slurried inethyl acetate, filtered and dried to providepentadecanoyl-l-hydroxybenzotriazole ester. L-aspartic acid 4-O-benzylester (0.2578 g, 1.156 mmol) was added to 2 mL of water and 2 mL oftetrahydrofuran followed by 1 mL of saturated sodium bicarbonatesolution and stirred until dissolved. A slurry ofpentadecanoyl-hydroxybenzotriazole (0.2798g, 0.758 mmol) in 5 mL ofwater and 5 mL of tetrahydrofuran was added and the reaction mixture wasstirred at ambient temperature overnight. The reaction mixture was thenpoured into 20 mL of water and acidified to about pH 1.0 with 6Nhydrochloric acid. The resulting precipitate was chilled, filtered anddried to afford 0.2792g. of pentadecanoyl-L-aspartic-acid-4-O-Benzylester in 79% yield. FAB-MS: m/z 448 (M+H)^(+,) 470 (M+Na)^(+,) 492(M+2Na-H)⁺.

5.12 Example 12: Synthesis Of Pentadecanoyl-L-Aspartic-Acid-4-O-BenzylEster

[0204] A mixture of pentadecanoyl-L-aspartic-acid-4-O-benzyl ester(0.2619 g, 0.5851 mmol), 1-hydroxybenzotriazole (0.0895 g, 0.5851 mmol),and dicyclohexylcarbodiimde (0.1205 g, 0.5851 mmol) in 5.0 mL oftetrahydrofuran was stirred at room temperature overnight. The reactionmixture was filtered and evaporated to dryness at reduced pressure. Theresulting oil was slurred in hexane to give 0.2933 g ofpentadecanoyl-L-aspartic-acid-4-O-benzyl-hydroxybenzotriazole ester as acrystalline product ( 88% yield).

5.13 Example 13: Synthesis Of The Benzyl Ester Of 100

[0205] A mixture of 6 (14.8 mg, 0.0162 mmol) and diisopropylethylamine(0.023 mL, 0.1319 mmol) was added to 0.5 mL of dimethylformamide andstirred at room temperature. Aliquots (0.20 mL) of a solution of thehydroxybenzotriazole ester ( 44.9 mg, 0.0794 mmol) prepared in Example12 were added to the solution of laspartomycin core peptide derivativeover 5 hours. Water was then added and the reaction mixture adsorbed ona 2.5×5.0 cm styrene-divinylbenzene resin column (ENVI™-Chrom P), andeluted with pH 7.2 phosphate buffer containing about 45% acetonitrile.Fractions containing the desired product were desalted and freeze driedto obtain 6.0 mg of white powder, the benzyl ester of 100. FAB-MS: m/z1339 (M+H)^(+,) 1361 (M+Na)³⁰ , and 1377 (M+K)⁺.

5.14 Example 14: Synthesis Of 100

[0206] A mixture of 3.0 mg of the benzyl derivative prepared in Example13, 11 mg of 5% palladium on carbon and 1.0 mL of methanol washydrogenated at atmospheric pressure overnight. The mixture was filteredthrough Celite, evaporated to dryness, slurried in water and lyophilizedto give 2.0 mg of 100. FAB-MS: m/z 1287 (M+K)^(+,) 1309 (M+K+Na-H)⁺.

5.15 Example 15: Synthesis Of Dihydro-Laspartomycin

[0207] A mixture of 21.3 mg of laspartomycin, 35 mg of 5% palladium oncarbon and 25 mL of methanol was hydrogenated at atmospheric pressureovernight (balloon technique). The mixture was filter through Celite,evaporated to dryness, slurried in water and lyophilized to give 19.4 mgof dihydro-laspartomycin. FAB-MS: m/z 1250 (M+H)^(+,) 1272 ( M+Na)⁺.

5.16 Example 16: Synthesis Of The Protected Derivative Of 54

[0208] t-Butoxycarbonyl-L-aspartic acid 4-O-butyl-1-hydroxybenzotriazoleester was prepared from t-butoxycarbonyl-4-O-butyl-L-aspartic acid,dicyclohexylcarbodiimide, and 1-hydroxybenzotriazole as described inExample 11 and used as described below.

[0209] A mixture of 6 (15.2 mg, 0.0167 mmol) and diisopropylethlyamine(0.025 mL, 0.1437 mmol) in 0.20 mL of dimethylformamide was stirred atroom temperature under nitrogen. A solution oft-butyoxycarbonyl-L-aspartic acid-4-O-t-butyl-hydroxybenzotriazole ester(0.030 mL aliquots) containing 0.0496 g (0.1218 mmol) of the activatedester in 0.20 mL was initially added and again after 0.50 hour. Theprogress of the reaction was followed by HPLC. When the reaction wascomplete the product was isolated as described in Example 13. Yield ofthe protected derivative of 54 was 9.0 mg, estimated 90% pure based onHPLC. FAB-MS: m/z 1182 (M+H)+, 1204 (M+Na)⁺.

5.17 Example 17: Synthesis Of 54

[0210]0.35 mL of trifluoroacetic acid was added to 6.9 mg of thecompound prepared in Example 16 and the solution was allowed to stand atroom temperature for 1.5 hours. Trifluoroacetic acid was removed and theresidue was lyophilized to afford 4.8 mg of 54 as the trifluoroacetatesalt. FAB-MS: m/z 1025 (M+H)^(+,) 1047 (M+Na)^(+,) 1063 (M+K)⁺.

5.18 Example 18: Sythesis Of 100

[0211] A solution of 54 (40 mg, 0.039 mmol) in 0.70 mL ofdimethylformamide containing 0.050 mL of duisopropylethylamine (0.288mmol) was stirred at room temperature and 0.35 mL of a solutioncontaining pentadecanoyl-1-hydroxybenzotriazole ester indimethylformamide, 41 mg (0.112 mmol) was added. After a 1.0 hour periodand after a 2.0 hour period an additional 0.17 mL and 0.25 mL of thissolution was respectively added. Duisopropylethylamine (0.025 mL) wasadded after 1.5 hours. The progress of the reaction was followed byHPLC. The product 100 was isolated by the general procedures describedin Example 13 except that the solvent to elute the product from thestyrene-divinylbenzene resin column (ENVI™-Chrom P) was 33% acetonitrilein water to provide 35 mg of a white powder, estimated 96% pure by HPLC.FAB-MS: mn/z 1 250(M+H)^(30 ,) 1272(M+Na)⁺.

5.19 Example 19: Synthesis Of 112

[0212] A mixture of p-dodecyloxybenzoic acid (0.3081g, 1.01 mmole),1-hydroxybenzotriazole (0.1559 g, 1.02 mmole), anddicyclohexylcarbodiimide (0.2091 g, 1.02 mmole) in 3 mL of DMF and 2 mLof THF was stirred at room temperature for 30 minutes. A 0.25 mL aliquotof this solution was added to a solution of 54 (0.060 g) in 0.25 mL ofDMF and stirred at room temperature. Additional aliquots of 0.20 mL ofthe activated ester were added after 40 minutes and 100 minutes. Thereaction mixture was quenched by addition of 5.0 mL of methanol and pHwas adjusted to pH 7 using pH paper by addition of 1.5 M ammoniumhydroxide (0.55 mL). This solution was applied to a 25×420 mm column ofSephadex LH-20 equilibrated in methanol. The sample was eluted withmethanol at about 0.8 mL/minutes and 7 ML fractions were collected.Product-containing fractions were pooled (retention volumes about 91-112mL) and methanol was removed under vacuum at 30-35° C. The residue wasdissolved in 8 mL distilled water, the pH adjusted to pH 7 by additionof a small volume of 1.5 M ammonium hydroxide, evaluated by HPLC, andfreeze dried. Yield: 12.8 mg of an off-white solid, 70% by HPLC (215 nmarea %); C₆₁H₉₂N₁₂O₂₀; FABMS: m/z 1336 (M+Na)⁺(calc. forC₆₁H₉₂N₁₂O₂₀+Na, 1336).

5.20 Example 20: Synthesis Of 114

[0213] Decanesulfonyl-L-phenylalanine (0.1167 g, 0.3158 mmole),1-hydroxybenzotriazole (0.0483 g, 0.3158 mmole), anddicyclohexylcarbodiimide (0.0650 g, 0.3158 mmole) in 0.86 mL of DMF wasstirred at room temperature for 45 minutes. A 0.20 mL aliquot of thissolution was added to a solution of 0.064 g of 54. The reaction mixturewas diluted with 4.0 mL of methanol and 0.30 mL of distilled water andthe pH was adjusted to about pH 7 using pH paper by addition of 1.5 Mammonium hydroxide. This solution was filtered using a membrane filter(Whatman GD/X, 13 mm) and applied to a Sephadex LH-20 column as inExample 19. Product-containing fractions were pooled and methanol wasremoved under vacuum at 30-35° C. The residue was further purified bypreparative HPLC on a Waters Delta-Pak C-18 column (25×220 mm, radialcompression system). The weak eluent was 10% isopropanol buffered with0.04 M ammonium phosphate (aqueous pH 7.2) and strong eluent was 50%isopropanol buffered with 0.008 M pH 7.2 buffer. The sample residue wasdissolved in weak eluent, applied to the column and eluted at roomtemperature at 10 mL/min using a gradient from 100% weak eluent to 20/80weak/strong eluents over 40 minutes. Product-containing fractions (asdetermined by analytical HPLC) were pooled and isopropanol was removedunder vacuum. The product solution was desalted by adsorption onto aconditioned styrene-divinylbenzene resin cartridge (0.5 g EnviChrom-P,Supelco, rinsed with 10 mL acetonitrile and 6 mL 12% acetonitrile).After sample application, the cartridge was rinsed with 5 mL ofsalt-free 12.5% acetonitrile and the product was stripped off thecartridge with 6 mL of 67% acetonitrile. The acetonitrile was removedunder vacuum at 30-35° C., the residue diluted to 10 mL with distilledwater then freeze dried. Yield: 10.0 mg white solid, 91% by HPLC (215 nmarea %); C₆₁H₉₃N₁₃O₂₁S; FABMS: m/z 1376 (M+H)^(+,) 1398(M+Na)^(+,) 1414(M+K)⁺(calc. for C₆₁H₉₃N₁₃O₂₁S+H, 1376).

5.21 Example 21: Synthesis Of 116

[0214] A mixture of p-decyloxybenzoic acid (0.1085 g, 0.390 mmole),diisopropylethylamine (0.068 mL, 0.390 mmole), and O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TOTU) (0.1274 g, 0.390 mmole) in 3.0 mL DMF was stirred at roomtemperature for 1.0 hours. A 0.27 mL aliquot of this solution was addedto a solution of 54 (0.050 g) in 1.0 mL DMF and stirred at roomtemperature. After 70 minutes, an additional 0.14 mL of the activatedester solution was added. After 90 minutes at room temperature thereaction mix was heated for 40 minutes at 50° C. The reaction mixturewas quenched by dilution with methanol and the pH adjusted to about pH 7with dilute ammonium hydroxide; the product was isolated on a SephadexLH-20 column as in Example 19. Product-containing fractions were pooledand methanol was removed under vacuum yielding 43 mg of a light yellowsolid. This product was further purified by low resolutionchromatography on a styrene-divinylbenzene resin cartridge (0.5 g,EnviChrom-P, Supelco). The LH-20 isolated solid was dissolved in 10 mLof 20% acetonitrile (0.10 M in ammonium phosphate (aqueous pH7.2)) andapplied to the open cartridge with gravity flow. The cartridge waseluted with stepwise increasing concentrations of acetontrile inammonium phosphate (about 0.05 M, pH7.2). The product was eluted with29% and 33% acetonitrile. Product-containing fractions were pooled,desalted and freeze dried as in Example 20. Yield: 23mg light tan solid,87% by HPLC (215nm area %); C₅₉H₈₈N₁₂O₂₀; FABMS: m/z 1285 (M+H)^(+,)1307(M+Na)^(+,) 1323 (M+K)⁺(calc. for C₅₉H₈₈N₁₂O₂₀+H, 1285).

5.22 Example 22: Synthesis Of 118

[0215] A mixture of dodecylisocyanate (0.0104 g, 0.043 mmole) and 54(0.044 g, 0.043 mmole) was stirred in DMF (0.60 mL). After 60 minutes atroom temperature, a second 0.0104 mL aliquot of the isocyanate was addedand stirred for 60 min. The reaction was quenched and product wasisolated on Sephadex LH-20 as in Example 19. Product-containingfractions were pooled, methanol removed under vacuum and the productfreeze dried from aqueous solution. Yield: 32mg of a white solid, 77% byHPLC (215 nm area %); C₅₅H₈₉N₁₃O₁₉; FABMS: m/z 1259 (M+Na)⁺(calc. forC₅₅H₈₉N₁₃O₁₉+Na, 1259).

5.23 Example 23: Synthesis Of 120

[0216] A mixture of dodecylchloroformate (0.010 mL) and 54 (0.035 g,0.034 mmole) in DMF (0.80 mL) was stirred at room temperature. Thereaction mixture was diluted with 4.0 mL methanol and the product wasisolated on a Sephadex LH-20 column as in Example 19. Methanol wasremoved under vacuum from the product-containing fractions; the residuewas dissolved in 10 mL of 14% acetonitrile (AcN) 0.10 M in ammoniumphosphate (aqueous pH7.2). This solution was desalted by application toa styrene-divinylbenzene resin cartridge (0.5 g, Supelco EnviChrom-P,conditioned with 10 mL AcN and 6 mL 14% AcN); the sample-loadedcartridge was rinsed with 6 mL salt-free 14% AcN and the product wasstripped off using 6 mL of salt-free 67% AcN. AcN was removed undervacuum and the product freeze dried out of aqueous solution. Yield: 11mg of a white solid, 81 % by HPLC (215 nm area %); C₅₅H₈₈N₁₂O₂₀; FABMS:m/z 1238 (M+H)^(+,) 1260(M+Na)^(+,) 1276 (M+K)⁺(calc. forC₅₅H₈₈N₁₂O₂₀+H, 1238).

5.24 Example 24: Synthesis Of 122

[0217] A mixture of 54 (0.0438 g) and hexadecylisocyanate (0.013 mL) in0.50 mL of DMF was stirred at room temperature. After 70 minutes asecond portion of the isocyanate was added. The reaction mixture wasquenched and the product isolated on a Sephadex LH-20 column as inExample 19. Methanol was removed under vacuum from theproduct-containing fractions yielding 29 mg of a yellow solid. Theproduct was further purified by low resolution reverse phasechromatography as in Example 21. The product was eluted with 40% AcN0.10 M in ammonium phosphate (aqueous pH7.2). This fraction was dilutedwith an equal volume of distilled water and desalted as in Example 23;18 mL of 67% AcN was necessary to elute the product. AcN was removedunder vacuum and the product was freeze dried from aqueous solution.Yield: 24 mg of an off-white solid, 83% by HPLC (215 nm area %);C₅₉H₉₇N₁₃O₁₉; FABMS: m/z 1292 (M+H)⁺.

5.25 Example 25: Synthesis Of 124

[0218] A mixture of 54 (0.0624 g) and tetradecylisothiocyanate (0.015mL) in 0.5 nIL DMF was stirred at room temperature for 1.0 hour. Asecond volume of the isothiocyanate (0.015 mL) was added and thereaction mixture was heated to 50° C. for several hours then raised to60° C. and an additional 0.015 mL of the reagent added and heated at 60°C. for 2 hours. A fourth volume of isothiocyanate (0.015 mL) was addedand the reaction stirred overnight at room temperature. The reactionmixture was quenched and the product isolated on a Sephadex LH-20 colunias in Example 19. Methanol was removed under vacuum from the appropriatepooled fractions yielding 43 mg of solid product. This product wasfurther purified on a 0.5 g styrene-divinylbenzene resin cartridge as inExample 21; product was eluted with 30% AcN 0.05 M in ammonium phosphate(aqueous pH7.2). Product-containing fractions were pooled, diluted withan equal volume of distilled water, desalted on a 0.5 g resin cartridgeas in Example 23, and freeze dried. Yield: 14 mg of a white solid, 75%by HPLC (215 nm area %); C₅₇H₉₃N₁₃O₁₈S; FABMS: m/z 1280 (M+H)^(+,)1302(M+Na)^(+,) 1318 (M+K)³⁰ (calc. for C₅₇H₉₃N₁₃O₁₈S+H, 1280).

5.26 Example 26: Synthesis Of 126

[0219] A mixture of p-dodecanamidobenzoic acid (0.1076 g, 0.34 mmole),O-[(ethoxycarbonyl) cyanomethyleneamino]-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TOTU) (0.1117 g, 0.34 mmole), anddiisopropylethylamine (0.059 mL, 0.34 mmole) in 1.0 mL of DMF wasstirred at room temperature for 1.0 hour. A 0.11 mL aliquot of thissolution of the activated ester was added to a solution of 54 (0.0624 g)in 0.5 mL of DMF and stirred at room temperature for 1.0 hour. A secondaliquot (0.10 mL) of activated ester solution was added after 1.0 hourand a third aliquot (0.10 mL) was added after 1.7 hour. The reactionmixture was quenched and the product was isolated on a Sephadex LH-20column as in Example 19. Methanol was removed under vacuum fromproduct-containing fractions, yielding 47 mg of solid which was furtherpurified on a 0.5 g styrene-divinylbenzene resin cartridge as in Example21. The product was eluted with 25% AcN 0.06 M in ammonium phosphate(aqueous pH7.2); fractions were desalted and freeze dried as in Example23. Yield: 24 mg of a white solid, 88% by HPLC (215 nm area %); C₆₁, H₉₁N₁₃O₂₀; FABMS: m/z 1326.5 (M+H)^(+,) 1348.6(M+Na)^(+,) 1364.5(M+K)⁺(calc. for C₆₁H₉₁N₁₃O₂₀ +H, 1326.6).

5.27 Example 27: Synthesis Of 128

[0220] A mixture of pentadecanoyl-L-phenylglycine-4-O-t-butyl-asparticacid (0.1834 g, 0.335 mmole), diisopropylethylamine (0.052 mL, 0.298mmole), and TOTU (0.0979 g, 10 0.298 mmole) in 1.0 mL of DMF was stirredat room temperature for 1.0 hour. A 0.25 mL aliquot of the activatedester solution was added to a solution of 54 (0.069 g) in 1.0 mL of DMF.After 75 minutes a second 0.50 mL portion of the activated estersolution was added. After 45 minutes the reaction mixture was quenchedas in Example 19, centrifuged and membrane filtered (Whatman GD/X, 13mm). Product was isolated from the filtrate on a Sephadex LH-20 columnas in Example 19; methanol was removed under vacuum fromproduct-containing fractions, yielding 72 mg of solid. The product wasfurther purified on a conditioned styrene-divinylbenzene resin cartridge(5.0 g EnviChrom-P). The LH-20 column residue was dissolved in 10 mL of20% AcN 0.08 M in ammonium phosphate (aqueous pH 7.2) and applied to theopen cartridge using gravity flow. The cartridge was eluted withstepwise increasing concentrations of AcN in ammonium phosphate (pH7.2);

[0221] product eluted with 45.5% AcN 0.04 M in ammonium phosphate.Product-containing fractions were pooled and diluted with an equalvolume of distilled water. The resin cartridge was rinsed with salt-free50% AcN (16 mL), 80% AcN (25 mL) and 20% AcN (20 mL) and the dilutedfraction pool was applied to the cartridge for desalting. The cartridgewas rinsed with salt-free 14% AcN (26 mL) and the product was strippedoff with 67% AcN (30 mL). AcN was removed from the fractions undervacuum and the aqueous solution was freeze dried. Yield: 25 mg of awhite solid, 74% by HPLC (215 nm area %); C₆₉H₁₀₇N₁₃O₂₀; FABMS: m/z1439(M+H)^(+,) 1461(M+Na)⁺, (calc. for C₆₉H₁₀₇N₁₃O₂₀+H, 1439).

5.28 Example 28: Synthesis Of 130

[0222] The product of Example 27 (24 mg) was dissolved in 1.0 mLtrifluoroacetic acid (TFA) and aged at room temperature for 1.0 hour.TFA was removed under vacuum to yield a small volume of abrownish-yellow oil. The oil was dissolved in 10 mL 20% AcN 5 0.08 M inammonium phosphate (aqueous pH 7.2) and decolorized and desalted on a0.5 g styrene-divinylbenzene cartridge as in Example 23. AcN was removedas an azeotrope under vacuum from the appropriate combined fractions andthe aqueous solution was freeze dried. Yield: 22 mg of a white solid,72% by HPLC (215 nm area %); C₆₅H₉₉N₁₃O₂₀; FABMS: m/z 1383 (M+H)^(+,)1405(M+Na)^(+,) 1421(+K)⁺, (calc. for C₆₅H₉₉N₁₃O₂₀ +H, 1383).

5.29 Example 29: Synthesis Of 164

[0223] N-pentadecanoyl-(O-benzyl)-L-glutamic acid was converted to the1-hydroxybenzotriazole ester using dicyclohexylcarbodiimide in the usualmanner and reacted with 6. The reaction mix was quenched and product wasisolated on a Sephadex LH-20 column as in Example 21. Methanol wasremoved from product-containing fractions under vacuum and the productwas further purified by low resolution chromatography on a 5 g resincartridge as in Example 27. Product was eluted with 46% AcN 0.025 M inanimonium phosphate (aqueous pH 7.2). After removal of AcN under vacuum,appropriate fractions were desalted and product isolated by freezedrying as in Example 27. Yield: 20 mg of a white solid, about 70% byHPLC (215 nm area %); C₆₅H₁₀₀N₁₂O₁₉; FABMS m/z 1353(M+H)^(+,)1375(M+Na)⁺, (calc. for C₆₅H₁₀₀N₁₂O₁₉ +H, 1353.7).

5.30 Example 30: Synthesis Of 166

[0224] The product of Example 29 (18 mg) was dissolved in 4.0 mL ofmethanol and 5% palladium on carbon (50 mg) was added. The mixture washydrogenated for 2.0 hours (balloon technique), membrane filtered(Whatman GD/X), and the filtrate evaporated to dryness. The residue wasdissolved in 10 mL of water by adjusting to pH 7 with dilute ammoniumhydroxide then freeze dried. Yield: 13 mg of a white solid, 64% by HPLC(215 nm area %); C₅₈H₉₄N₁₂O₁₉; FABMS m/z 1264 (M+H)^(+,) 1286 (M+Na)⁺,and 1308 (M+2Na)⁺, (calc. for C₅₈H₉₄N₁₂O₁₉+H, 1263.7).

5.31 Example 31: Synthesis Of 146

[0225] A solution of N-pentadecanoyl-L-phenylalanine (76.8 mg, 0.197mmole), 1-hydroxybenzotriazole (30.1 mg, 0.197 mmole), anddicyclohexylcarbodiimide (40.6 mg, 0.197 mmole) in 1.0 mL DMF wassonicated and stirred at room temperature for 45 minutes. A 0.25 mLaliquot of this solution was added to 54 (48 mg) in 0.20 mL of DMF andstirred at room temperature for 70 minutes. A second 0.050 mL aliquot ofactivated ester solution was added and stirred for 40 minutes. Thereaction mixture was diluted with 4 mL of methanol, aged 10 minutes atroom temperature and filtered through a membrane (Gelman Acro LC13). Theproduct was isolated on a Sephadex LH-20 column as in Example 21. Theproduct was further purified on a 5 g resin cartridge as in Example 27and eluted from the resin with 46% AcN 0.025 M in ammonium phosphate(pH7.2). The product was desalted and freeze dried as in Example 27.Yield: 10 mg of a white solid, 96% by HPLC(215nm area %); C₆₆H₁₀₁N₁₃O₂₀; FABMS m/z 1396 (M+H)^(+,) 1418(M+Na)⁺, and 1434 (M+K)⁺, (calc.for C₆₆H₁₀₁N₁₃O₂₀+H, 1396.7).

5.32 Example 32: Synthesis Of 146

[0226] N-pentadecanoyl-D-phenylalanine was converted to the activatedester as described in Example 31 for the L-isomer and reacted with 54.The reaction mixture was quenched and product isolated as in Example 31;methanol was removed under vacuum from product-containing fractionsyielding 32 mg of solid. The product was further purified and isolatedas in Example 31. Yield: 17 mg of a white solid, 97% byHPLC (215 nm area%); C₆₆H₁₀₁N₁₃ O₂₀; FABMS m/z 1397 (M+H)^(+,) 1419(M+Na)⁺(calc. forC₆₆H₁₀₁N₁₃O₂₀+H, 1396.7).

5.33 Example 33: Sythesis Of 152

[0227] A solution of α-N-pentadecanoyl-F-benzyloxycarbonyl-L-lysine (101mg, 0.20 mmole), 1-hydroxybenzotriazole (31 mg, 0.20 mmole), anddicyclohexylcarbodiimide (41 mg, 0.20 mmole) in 1.5 mL DMF was stirredat room temperature for 45 minutes. A 0.45 mL aliquot of this solutionwas added to a solution of 54 (52.5 mg) in 0.30 mL of DMF and stirred atroom temperature for 60 minutes. The reaction mixture was diluted with5.0 mL of methanol, filtered through a membrane (Gelman Acro LC13) andthe product isolated on a Sephadex LH-20 column as in Example 21. Thesolid residue was further purified on a 5 g resin cartridge as inExample 27; the product was eluted from the resin with 46% AcN 0.025 Min ammonium phosphate (pH 7.2). Appropriate fractions were desalted andproduct isolated by freeze drying as in Example 25. Yield: 7 mg of awhite solid, 72% by HPLC (215nm area %); C₇₁H₁₁₀N₁₄O₂₂; FABMS: m/z 1512(M+H)^(+,) 1534(M+Na)⁺, and 1550 (M+K)⁺, (calc. for C₇₁H₁₁₀N₁₄O₂₂+H,1511.8).

5.34 Example 34: Synthesis Of 154

[0228] To a solution of 3.8 mg of 152 in 2.0 mL of methanol was added5.5 mg of 10% palladium on carbon. The mixture was stirred andhydrogenated (balloon technique) at room temperature for 1.0 hours. Thecatalyst was removed by filtration and the methanol was evaporated atreduced pressure. The residue was dissolved in water and freeze dried.Yield: 2.5 mg, 72% by HPLC (215 nm area %); C₆₃H₁₀₄N₁₄O₂₀; FABMS m/z1400 (M+Na)⁺, (calc. for C₆₃H₁₀₄N₁₄O₂₀+Na, 1399.7).

5.35 Example 35: Synthesis Of 178

[0229] A mixture of pentadecanoyl-(O-t-butyl)-L-tyrosine (56 mg, 0.121mmole), 1-hydroxybenzotriazole (19.8 mg, 0.129 mmole), anddicyclohexylcarbodiimide (25 mg, 0.121 mmole) in 0.43 mL of DMF wasstirred at room temperature for 45 minutes. A 0.30 mL aliquot of thissolution was added to 58 mg of 54 in 0.25 mL of DMF and stirred at roomtemperature. After 1.0 hours the remainder of the activated ester wasadded and stirred for 40 minutes. The reaction mix was quenched andproduct isolated on a Sephadex LH-20 column as in Example 21. Methanolwas removed under vacuum from product-containing fractions yielding 25mg solid residue which was further purified and isolated on a 5 g resincartridge as in Example 27. Yield: 14 mg, 93% by HPLC (215 nm area %);C₇₀H ₁₀₉N₁₃O₂₁; FABMS m/z 1468.5 (M+H)^(+,) 1490.5 (M+Na)^(+,) 1506.3(M+K)⁺, (calc. for C₇₀H₁₀₉N₁₃O₂₁+H, 1468.8 ).

5.36 Example 36: Synthesis Of 180

[0230] A solution of 178 from Example 35 (11 mg) in 1.5 mL of 95%trifluoroacetic acid (TFA) was stirred at room temperature for 70minutes. HPLC indicated the reaction was complete. TFA was removed witha stream of dry nitrogen and the residue was dried in vacuo overpotassium hydroxide. The resulting dry solid was dissolved in a few mLof water by adding one drop of 3% ammonium hydroxide and freeze dried.Yield: 10 mg, 70% by HPLC (215 nm area %); C₆₆H₁₀₁N₁₃O₂₁; FABMS m/z 1413(M+H)^(+,) 1435 (M+Na)⁺, ( calc. for C₆₆H₁₀₁N₁₃O₂₁+H, 1412.7).

5.37 Example 37: Synthesis Of 139

[0231] N-Hexadecylsulfonyl-(O-t-butyl)-L-aspartic acid (189 mg, 0.395mmole), 1-hydroxybenzotriazole (55.1 mg, 0.395 mmole) anddicyclohexylcarbodiimide (82.5 mg, 0.395 mmole) in 0.50 mL DMF wasstirred at room temperature for 45 minutes. A 0.050 mL aliquot of thissolution was added to the tetrabutylammonium salt of 6 (25 mg) in 0.20mL of DMF and stirred at room temperature for 60 minutes. The reactionmixture was quenched by dilution with 8 mL of 25% acetonitrile (AcN)0.12 M in ammonium phosphate (pH 7.2), aged at room temperature, thenfiltered through a membrane (Whatman GD/X). The product was isolatedfrom the filtrate by low resolution reverse phase chromatography on a 5g styrene-divinylbenzene resin cartridge (25×45 mm, SupelcoEnviChrom-P). The sample-loaded cartridge was eluted with stepwiseincreasing concentrations of AcN in sodium phosphate (aqueous pH 6.9);product was eluted with 57% AcN 0.010 M in pH 6.9 buffer.Product-containing fractions were pooled, diluted with an equal volumeof distilled water and then desalted on the same 5g resin cartridgewhich had been rinsed with 75% and 25% AcN. The diluted fraction poolwas applied to the cartridge, which was rinsed with 16 mL 25% AcN 0.125M in ammonium phosphate (pH 7.2), then 24 mL salt-free 25% AcN. Theproduct was stripped from the cartridge with 48 mL 67% AcN. AcN wasremoved under vacuum from the strip fraction and the aqueous solutionwas freeze dried to give the desired product. Yield: 4.7 mg of a whitesolid, 69% by HPLC (215 nm area %); C₆₂H₁₀₄N₁₂O₂₀S; FABMS m/z 1392(M+Na)⁺. (calc. for C₆₂H₁₀₄N₁₂O₂₀S+Na, 1391.7).

5.38 Example 38: Synthesis Of 140

[0232] A solution of 139 (4.7 mg) in 0.50 mL of 95% trifluoroacetic acid(TFA) was stirred at room temperature for 30 minutes. TFA was removedwith a stream of dry nitrogen and the residue was triturated witht-butylmethyl ether and centrifuged. Excess ether was removed and theresulting solid was dissolved in 1.5 mL of water by adding 1 drop of 3%ammonium hydroxide, then freeze dried. Yield: 2.5 mg of solid, 63% byEPLC (215 nm area %); C₅₈H₉₆N₁₂O₂₀S; FABMS m/z 1313 (M+H)^(+,) 1335(M+Na)⁺, (calc. for C₅₈H₉₆N₁₂O₂₀S+H, 1313.6)

5.39 Example 39: Synthesis Of 155

[0233]α-N-Pentadecanoyl-(N-t-butyloxycarbonyl)-L-tryptophyl-(N-trityl)-L-asparaginewas converted to the 1-hydroxybenzotriazole activated ester usingdicyclohexylcarbodiimide by the usual protocol (DMF as solvent) andreacted with 6 (63.6 mg) in 0.4 mL DMF for 80 minutes at roomtemperature. The reaction mixture was quenched and product isolated on aSephadex LH-20 column as in Example 21. The product was further purifiedon a 5 g resin cartridge as in Example 27; product was eluted with 66%AcN 0.005 M in ammonium phosphate (pH 7.2). Product-containing fractionswere desalted and freeze dried as in Example 27. Yield: 22 mg of a whitesolid, 79% by HPLC (220 nm area %); C₉₂H₁₂₅N₁₅O₂₁; FABMS m/z 1777(M+H)^(+,) 1779 (M+Na)⁺, (calc. for C₉₂H₁₂₅N₁₅O₂₁+H, 1776.9).

5.40 Example 40: Synthesis Of 156

[0234] The protected derivative 155 from Example 39 was treated with 1.5mL of 95% trifluoroacetic acid for 30 minutes at room temperature. Thesolution was processed as described in Example 38. Theether-precipitated solid was dissolved in 6 mL of water, the pH wasadjusted to 4.0 with dilute ammonium hydroxide and then heated at 50° C.for thirty minutes. The solution pH was adjusted to about pH 7.0 withammonium hydroxide and then freeze dried. Yield: 12 mg, 79% by HPLC (215nm area %); C₆₈H₁₀₃N₁₅O₁₉; FABMS: m/z 1435 (M+H)^(+,) 1457 (M+Na)⁺,(calc. for C₆₈H₁₀₃N₁₅O₁₉+H, 1434.8).

5.41 Example 41: Synthesis Of 133

[0235] A solution of N-pentadecanoyl-L-tryptophyl-(O-t-butyl)-L-asparticacid (50 mg, 0.083 mmole), 1-hydroxybenzotriazole (14.6 mg, 0.095 mmole)and dicyclohexylcarbodiimide (17.2 mg, 0.083 mmole) in 0.80 mL DMF wasstirred at room temperature for 1.0 hours. A 0.050 mL aliquot of thissolution was added to a solution of about 80 mg of 6 in 0.20 mL of DMF.An additional 0.050 mL of the activated ester was added after 100minutes and stirred for an additional 50 minutes. The reaction mixturewas diluted with 6 mL of 33% AcN 0.17 M in ammonium phosphate (pH 7.2)then filtered through a membrane(Whatman GD/X). Two diastereomericproducts were isolated by preparative HPLC on a Waters Delta-Pak C18column (25×110 mm). It is assumed that the epimerization ocurred at thea carbon of the aspartic acid residue in the linking side chain. Thecolumn was eluted at 10 mL/min at room temperature using a lineargradient from 38% AcN 0.022 M in sodium phosphate (pH 6.9) to 54% AcN0.016 M in sodium phosphate (pH 6.9) over 60 minutes. Appropriatefractions were pooled for each of the two diastereomeric products; AcNwas removed under vacuum and the fractions were desalted on a 3 g resincartridge as in Example 37. AcN was removed under vacuum from the stripfractions and the aqueous solutions were freeze dried. Yields: D-diastereomer: 18 mg of a white solid, 88% by HPLC (215nm area %);C₇₂H₁₁₀N₁₄O₂₀; FABMS m/z 1492 (M+H)^(+,) 1514(M+Na)⁺, (calc. forC₇₂H₁₁₀N₁₄O₂₀+H, 1491.8). L-diastereomer: 30 mg of a white solid, 95% byHPLC (215 nm area %); C₇₂H₁₁₀N₁₄O₂₀ FABMS m/z 1492 (M+H)^(+,)1513(M+Na)⁺, (calc. for C₇₂H₁₁₀N₁₄O₂₀+H, 1491.8).

5.42 EXAMPLE 42: Synthesis Of 134

[0236] A solution of the presumed L-isomer (30 mg) of 133 in 0.50 mL oftrifluoroacetic acid (TFA) was stirred at room temperature under argonfor 1.0 hours. TFA was removed with a stream of dry nitrogen. Theresidue was stored over potassium hydroxide in vacuo overnight thendissolved in t-butanol and freeze dried. Yield: 28 mg of solid, 90% byHPLC (215 nm area %); C₆₈H₁₀₂N₁₄O₂₀; FABMS m/z 1435 (M+H)^(+,) 1457(M+Na)^(+,) 1473 (M+K)⁺, (calc. for C₆₈H₁₀₂N₁₄O₂₀+H, 1435.7).

5.43 Example 43: Synthesis Of 134

[0237] A solution of the presumed D-isomer (18 mg) of 133 in 0.50 mL oftrifluoroacetic acid (TFA) was stirred at room temperature under argonfor 1.0 hours. TFA was removed with a stream of dry nitrogen. Theresidue was stored over potassium hydroxide in vacuo overnight thendissolved in t-butanol and freeze dried. Yield: 17 mg of solid, 90% byHPLC (215 nm area %); C₆₈H₁₀₂N₁₄O₂₀; FABMS m/z 1435 (M+H)^(+,) 1457(M+Na)^(+,) 1473 (M+K)⁺, (calc. for C₆₈H₁₀₂N₁₄O₂₀+H, 1435.7). Thiscompound did not exhibit any in vitro biological activity.

5.44 Example 44: Synthesis Of 143

[0238]N-Pentadecanoyl-(N-t-butyloxycarbonyl)-L-tryptophyl-(N-trityl)-L-asparaginyl-(O-t-butyl)-L-aspartic acid was converted to the activated ester in DMFusing 1-hydroxybenzotriazole and dicyclohexylcarbodiimide. This wasreacted with 6 (41 mg) in 0.20 mL DMF. The reaction mixture was quenchedas in Example 41 and the product was isolated on a 5 g resin cartridgeas in Example 27. The product was eluted with 67% AcN (no buffer salt).AcN and water were removed under vacuum from the product-containingfractions. Yield: 32 mg of a white solid, 71% by HPLC (220 nm area %);C₁₀₀H₁₃₈N₁₆O₂₄.

5.45 Example 45: Synthesis Of 144

[0239] The protected peptide derivative prepared in Example 44 (30 mg)was treated with 1.5 mL of 95% TFA and worked up as in Example 40. Thehydrolyzed solution was freeeze dried, yielding 13 mg of an off-whitesolid. This product was dissolved in 1.0 mL of methanol and furtherpurified on a Sephadex LH-20 column (25×80 mm, equilibrated in methanol)which was eluted with methanol. Methanol was removed fromproduct-containing fractions and product was freeze dried from anaqueous solution and adjusted to pH7 with dilute ammonium hydroxide.Yield: 9 mg of a white solid, 80% by HPLC (220 nm area %); C₇₂H₁₀₈N₁₆O₂₂FABMS m/z 1551(M+H)^(+,) 1571(M+Na)^(+,) 1588(M+K)⁺, (calc. forC₇₂H₁₀₈N₁₆O₂₂+H, 1549.8).

5.46 Example 46: Synthesis Of 142

[0240] A solution of 54 (38 mg) in 0.20 ml of DMF was treated with 0.20ml of a DMF solution containing 1.1 equivaents ofN-pentadecanoyl-L-alanine 1-hydroxybenzotriazole activated ester(prepared in the usual manner) and stirred at room temperature. Afterone hour an additional 0.20 ml of the activated ester solution wasadded. Forty-five minutes after the second addition the reaction wasquenched by pouring into 50 ml of water. The resulting mixture wasadjusted to pH 9.5, 94 mg of Ca₂Cl was added, and the resulting solutionwas extracted with 50 ml 1-butanol followed by another 25 milliliters.The combined 1-butanol extracts were evaporated with addition of water(azeotrope) to an aqueous solution which was freeze dried to obtain 20mg of white powder. This material contained some residual HOBT and waschromatographed on Sephadex LH-20 with MeOH by the standard procedure(see Example 21) to obtain 14 mg of white powder, 89% pure by HPLC(215nm area %), C₆₀H₉₇N₁₃O₂₀; FABMS m/z 1320 (M+H)^(+,) 1342 (M+Na)⁺, (calc.for C₆₀H₉₇N₁₃O₂₀+H, 1320.7).

5.47 Example 47: Mass Spectral Data For Peptide Intermediates Used ToSynthesize Laspartmomycin Derivatives

[0241] Listed below are mass spectral data for various peptideintermediates used to synthesize laspartomycin derivatives in thepreceding Examples. The peptide derivatives were listed below weregenerally prepared by the activated ester method as in Example 20.Compound* FABMS N-pentadecanoyl-L-phenylglycyl-L-(O-t-butyl)- 547(M +H)⁺ aspartic acid N-pentadecanoyl-L-phenylglycine methyl ester 90(M +H)⁺ N-pentadecanoyl-L-phenylglycine 376(M + H)⁺, 398(M + Na)⁺ (Preparedby hydrolysis of ester) N-pentadecanoyl-L-(O-benzyl)-glutamic acid426(M + H)⁺ N-pentadecanoyl-L-(O-t-butyl)-tyrosine methyl ester 476(M +H)⁺ N-pentadecanoyl-L-(O-t-butyl)-tyrosine 462(M + H)⁺ (Prepared byhydrolysis of ester) N-pentadecanoyl-D-phenylalanine 390(M + H)⁺N-pentadecanoyl-L-phenylalanine 390(M + H)⁺α-N-pentadecanoyl-ε-benzyloxycarbonyl-L-lysine 505(M + H)⁺N-pentadecanoyl-L-alanine 314(M + H)⁺, 336(M + Na)⁺N-benzyloxycarbonyl-L-tryptophyl-L-(O-t-butyl) 510(M + H)⁺ -asparticacid L-tryptophyl-L-(O-t-butyl)-aspartic acid 376(M + H)⁺ (Prepared byhydrogenolysis of CBZ derivative)

5.9 MIC Data for Laspartomycin Derivatives

[0242] MIC values were determined by microliter serial dilution usingStaphlococcus aureus strain Smith as the assay organism, which was grownin Mueller-Hinton broth with and without CaCl₂. Name w/o CaCl₂ w/CaCl₂Daptomycin 1 0.5 Aspartocin 2 1 Zaomycin 10 1 Laspartomycin 16 2 146 164 134 (L) 32 8 134 (D) >64 >64 138 >64 >64 142 >64 >64 144 >64 16136 >64 >64 182 >64 >64 152 >64 8 154 >64 16 146 16 4 156 >64 8 178 >648 180 >64 8 164 >128 >128 166 >128 64 168 128 64 184 >128 >128 174 >128128 186 >128 >128 112 4 2 116 16 1.3 118 32 4 120 32 8 122 4 2

[0243] Although the foregoing invention has been described in somedetail to facilitate understanding, it will be apparent that certainchanges and modifications may be practiced within the scope of theappended claims. Accordingly, the present embodiments are to beconsidered as illustrative and not restrictive, and the invention is notto be limited to the details given herein, but may be modified withinthe scope and equivalents of the appended claims.

We claim:
 1. A method for making a laspartomycin core peptide, salt orhydrate thereof, comprising the steps of: culturing the microorganismStreptomyces viridochromogenes, ssp. komabensis (ATCC 29814) in aculture medium; isolating laspartomycin from the culture medium; andcleaving a lipophilic fragment from laspartomycin, thereby yielding thelaspartomycin core peptide.
 2. The method of claim 1 further includingthe step of isolating the laspartomycin core peptide.
 3. The method ofclaim 1 in which the culturing step is carried out at a temperature inthe range of about 24° C. to about 34° C.
 4. The method of claim 3 inwhich the temperature is in the range of about 27° C. to about 29° C. 5.The method of claim 1 in which the microorganism is removed from theculture medium prior to isolating laspartomycin.
 6. The method of claim5 in which the culture medium is acidified prior to removing themicroorganism.
 7. The method of claim 6 in which the culture medium isacidified to a pH in the range of about 2.0 to about 3.0.
 8. The methodof claim 7 in which the microorganism is removed via centrifugation andsuspended in water, thereby providing an aqueous suspension.
 9. Themethod of claim 8 in which the pH of the aqueous suspension is adjustedto a basic pH.
 10. The method of claim 8 in which a divalent cationconcentration of the aqueous suspension is adjusted to between about4mmol/l to about 10 mmol/l and the pH of the aqueous suspension isadjusted to a basic pH.
 11. The method of claim 9 or 10 in which theadjusted pH is in the range of about pH 8.0 to about pH 9.0.
 12. Themethod of claim 10 in which the divalent cation is selected from thegroup consisting of Ca²⁺, Mg²⁺and Zn⁺².
 13. The method of either ofclaim 9 or claim 10, in which laspartomycin is extracted into organicsolvent, thereby providing an organic solvent extract of laspartomycin.14. The method of claim 13 further comprising: acidifying the organicsolvent extract of laspartomycin; extracting laspartomycin into aqueoussolution; extracting laspartomycin into organic solvent; extractinglaspartomycin into aqueous solution; and concentrating the aqueoussolution to provide a salt of laspartomycin.
 15. The method of claim 14in which the organic solvent is 1-butanol.
 16. The method of claim 14,wherein the salt of laspartomycin is extracted into aqueous solution bywashing the organic solvent extract of laspartomycin with aqueous basesolution.
 17. The method of claim 14, wherein laspartomycin is extractedinto organic solvent by acidifying the aqueous solution of the salt oflaspartomycin.
 18. The method of claim 14, further comprising:dissolving the salt of laspartomycin in aqueous acid solution;extracting laspartomycin into organic solvent; and removing the organicsolvent to provide laspartomycin.
 19. The method of claim 1 in which thelipophilic fragment is cleaved with an enzyme.
 20. The method of claim19 in which the enzyme is a deacylase.
 21. The method of claim 1 inwhich the cleavage step further comprises: culturing a microorganismcapable of producing a deacylase in a culture medium; and contactinglaspartomycin with the culture medium.
 22. The method of claim 21 inwhich the microorganism is Actinoplanes utahensis (NRRL 12052).
 23. Themethod of claim 22 in which laspartomycin is contacted with the culturemedium for about 16 hours at about 29° C.
 24. The method of claim 22 inwhich laspartomycin is contacted with the culture medium for about 4hours at about 29° C.
 25. The method of claim 23 in which thelaspartomycin core peptide has the structure:

or a salt or hydrate thereof
 26. The method of claim 24 in which thelaspartomycin core peptide has the structure:

or a salt or hydrate thereof.
 27. The laspartomycin core peptideproduced by the method of any one of claims 1, 23 and
 24. 28. Alaspartomycin core peptide derivative according to structural formula(I): Y ¹—L—X¹—N(R¹)—R  (I) or a salt or hydrate thereof, wherein either:(i) Y¹—L—X¹ taken together is hydrogen; or (ii) Y¹ is a linking group; Lis a linker; X¹ is selected from the group consisting of —CO——SO₂—,—CS—, —PO—, —OPO—, —OC(O)—, —NHCO— and —NR¹CO—; N is nitrogen; R¹ isselected from the group consisting of hydrogen, (C₁-C₁₀) alkyloptionally substituted with one or more of the same or different R²groups, (C₁-C₁₀) heteroalkyl optionally substituted with one or more ofthe same or different R² groups, (C₅-C₁₀) aryl optionally substitutedwith one or more of the same or different R² groups, (C₅-C₁₅) arylaryloptionally substituted with one or more of the same or different R²groups, (C₅-C₁₅) biaryl optionally substituted with one or more of thesame or different R² groups, five to ten membered heteroaryl optionallysubstituted with one or more of the same or different R² groups,(C₆-C₁₆) arylalkyl optionally substituted with one or more of the sameor different R² groups and six to sixteen membered heteroarylalkyloptionally substituted with one or more of the same or different R²groups; each R² is independently selected from the group consisting of—OR³, —SR³, —NR³R³, —CN, —NO₂, —N₃, —C(O)OR³, —C(O)NR³R³, —C(S)NR³R³,—C(NR³)NR³R³, —CHO, —R³CO, —SO₂R³, —SOR³, —PO(OR³)₂, —PO(OR³), —CO₂H,—SO₃H, —PO₃H, halogen and thihalomethyl; each R³ is independentlyselected from the group consisting of hydrogen, (C₁-C₆) alkyl, (C₅-C₁₀)aryl, 5-10 membered heteroaryl, (C₆-C₁₆) arylalkyl and six to sixteenmembered heteroarylalkyl; and R is the core cyclic peptide oflaspartomycin.
 29. The laspartomycin core peptide derivative of claim 28wherein R has the structure:


30. The laspartomycin core peptide derivative of claim 29 in which Y¹ isselected from the group consisting of —NHR¹, —NH₂, —OH, —SH, —PH,halogen, —CHO, —R¹CO, —SO₂H, —PO₂H, —N₃, —CN, —CO₂H, —SO₃H, —PO₃H,—PO₂(OR¹)H, —CO₂R¹, —SO₃R¹, and —PO(OR¹)₂.
 31. The laspartomycin corepeptide derivative of claim 30 in which R¹ is hydrogen.
 32. Thelaspartomycin core peptide derivative of claim 31 in which Y¹ isselected from the group consisting of —SH, H₂N—, —OH, —CO₂H and —CO₂R,X¹ is carbonyl and L is selected from the group consisting of:

or a salt or hydrate thereof, wherein: n is 0, 1, 2or 3; each S¹ isselected from the group consisting of hydrogen, (C₁- C₁₀) alkyloptionally substituted with one or more of the same or differentRgroups, (C₁-C₁₀) heteroalkyl optionally substituted with one or more ofthe same or different R⁴ groups, (C₅-C₁₀) aryl optionally substitutedwith one or more of the same or different R⁴ groups, (C₅-C₁₅) arylaryloptionally substituted with one or more of the same or different R⁴groups, (C₅-C₁₅) biaryl optionally substituted with one or more of thesame or different R⁴ groups, five to ten membered heteroaryl optionallysubstituted with one or more of the same or different R⁴ groups,(C₆-C₁₆) arylalkyl optionally substituted with one or more of the sameor different R⁴ groups and six to sixteen membered heteroarylalkyloptionally substituted with one or more of the same or different R⁴groups; each R⁴ is independently selected from the group consisting of—OR⁵, —SR⁵, —NR⁵R⁵, —CN, —NO₂, —N₃, —C(O)OR⁵, —C(O)NR⁵R⁵, —C(S)NR⁵R⁵,—C(NR⁵)NR⁵R⁵, —CHO, —R⁵CO, —SO₂R⁵, —SOR⁵, —PO(OR⁵)₂, —PO(OR⁵), —CO₂H,—SO₃H, —PO₃H, halogen and trihalomethyl; each R⁵ is independentlyselected from the group consisting of hydrogen, (C₁-C₆) alkyl, (C₅-C₁₀)aryl, 5-10 membered heteroaryl, (C₆-C₁₆) arylalkyl and six to sixteenmembered heteroarylalkyl; and each K is independently selected from thegroup consisting of oxygen, nitrogen, sulfur and phosphorus.
 33. Thelaspartomycin core peptide derivative of claim 32 in which each S¹ isindependently a side-chain of a genetically encoded α-amino acid. 34.The laspartomycin core peptide derivative of claim 32 in which Y¹ isH₂N— and L is:


35. The laspartomycin core peptide derivative of claim 34 in which eachS¹ is independently a side-chain of a genetically encoded α-amino acid.36. The laspartomycin core peptide derivative of claim 35 in which n is0 and S¹ is —CH₂C(O)OH or a salt or hydrate thereof.
 37. Thelaspartomycin core peptide derivative of claim 35 in which n is 1 and S¹is —CH₂CO₂H or a salt or hydrate thereof and S² is —CH₂-indol-2-yl. 38.The laspartomycin core peptide derivative of claim 28 in which Y¹—L—X¹taken together is hydrogen and R¹ is hydrogen.
 39. A method for making alaspartomycin core peptide derivative comprising covalently attaching alinker moiety to a laspartomycin core peptide.
 40. A method of making aantimicrobial laspartomycin derivative comprising: covalently attachinga linker moiety to a laspartomycin core peptide, thereby providing alaspartomycin core peptide derivative; and covalently attaching alipophilic group to the laspartomycin core peptide derivative to yield aantimicrobial laspartomycin derivative.
 41. The method of claim 40further including the step of isolating the antimicrobial laspartomycinderivative.
 42. The method of claim 40 in which the laspartomycin corepeptide is provided by the method of any one of claims 1, 23 and
 24. 43.The method of claim 40 in which the laspartomycin core peptide is acompound according to any one of claims 36 and
 38. 44. A method ofmaking a antimicrobial laspartomycin derivative comprising: covalentlyattaching a lipophilic group to a linker, thereby providing alipophilic-linker group; and covalently attaching the lipophilic-linkergroup to the laspartomycin core peptide derivative thereby yielding aantimicrobial laspartomycin derivative.
 45. The method of claim 44further including the step of isolating the antimicrobial laspartomycinderivative.
 46. The method of claim 44 in which the laspartomycin corepeptide is provided by the method of any one of claims 1, 23 and
 24. 47.The method of claim 44 in which the laspartomycin core peptide is acompound according to any one of claims 36 and
 38. 48. The laspartomycinderivative provided by the method of any one of claims 40 and
 44. 49. Anisolated antimicrobial laspartomycin derivative according to structuralformula (II): Y ²—(X²—X³)—L—X¹—N (R¹)—R  (II) or an pharmaceuticallyacceptable salt or hydrate thereof, wherein: Y² is a lipophilic group;X¹ is selected from the group consisting of —CO—, —SO₂—, —CS—, —PO—,—OPO—,—OC(O)—, —NHCO— and —NR¹CO—; X² is a linked group; X³ is a linkedgroup; L is a linker; N is nitrogen; R¹ is selected from the groupconsisting of hydrogen, (C₁-C₁₀) alkyl optionally substituted with oneor more of the same or different R² groups, (C₁-C₁₀) heteroalkyloptionally substituted with one or more of the same or different R²groups, (C₅-C₁₀) aryl optionally substituted with one or more of thesame or different groups, (C₅-C₁₅) arylaryl optionally substituted withone or more of the same or different R² groups, (C₅-C₁₅) biaryloptionally substituted with one or more of the same or different R²groups, five to ten membered heteroaryl optionally substituted with oneor more of the same or different R² groups, (C₆-C₁₆) arylalkyloptionally substituted with one or more of the same or different R²groups and six to sixteen membered heteroarylalkyl optionallysubstituted with one or more of the same or different R² groups; each R²is independently selected from the group consisting of —OR³, —SR³,—NR³R³, —CN, —NO₂, —N₃, —C(O)OR³, —C(O)NR³R³, —C(S)NR³R³, —C(NR³)NR³R³,—CHO, —R³CO, —SO₂R³, —SOR³, —PO(OR³)₂, —PO(OR³), —CO₂H, —SO₃H, —PO₃H,halogen and trihalomethyl; each R³ is independently selected from thegroup consisting of hydrogen, (C₁-C₆) alkyl, (C₅-C₁₀) aryl, 5-10membered heteroaryl, (C₆-C₁₆) arylalkyl and six to sixteen memberedheteroarylalkyl; and R is the core cyclic peptide of laspartomycin. 50.The laspartomycin derivative claim 49 in which R has the structure:


51. The laspartomycin derivative of claim 50 in which (X²—X³) takentogether are selected from the group consisting of —C(O)O—, —O(O)C—,—CONH—, —NHCO—, —CONR¹—, —NR¹CO—, —C(O)S—, —S(O)C—, —OS₂—, —S(O₂)O—,—NHSO₂—, —NR¹SO₂, —S(O₂)NH—, —S(O₂)NR¹—, C(S)NH—, —NHC(S)—, —NHP(O)—,—P(O)NH—, —OP(O)—, —P(O)O—, —SP(O)—, —P(O)S—, —OC(O)NH—, —NHC(O)O—,—OC(O)NR¹—, —NR¹C(O)O—, —OC(O)O—, —NHC(O)NH—, —NHC(O)NR¹—, —NR¹C(O)NH—and NR¹C(O)NR¹.
 52. The laspartomycin derivative of claim 51 in which R¹is hydrogen.
 53. The laspartomycin derivative of claim 52 in which X¹ is—CO— or —SO₂—, and L is selected from the group consisting of:

or a pharmaceutically acceptable salt or hydrate thereof, wherein: n is0, 1, 2or 3; each S¹ is selected from the group consisting of hydrogen,(C₁-C₁₀) alkyl optionally substituted with one or more of the same ordifferent R⁴ groups, (C₁-C₁₀) heteroalkyl optionally substituted withone or more of the same or different R⁴ groups, (C₅-C₁₀) aryl optionallysubstituted with one or more of the same or different R⁴ groups,(C₅-C₁₅) arylaryl optionally substituted with one or more of the same ordifferent R⁴ groups, (C₅-C₁₅) biaryl optionally substituted with one ormore of the same or different R⁴ groups, five to ten membered heteroaryloptionally substituted with one or more of the same or different R⁴groups, (C₆-C₁₆) arylalkyl optionally substituted with one or more ofthe same or different R⁴ groups and six to sixteen memberedheteroarylalkyl optionally substituted with one or more of the same ordifferent R⁴ groups; each R⁴ is independently selected from the groupconsisting of —OR⁵, —SR⁵, —NR⁵R⁵, —CN, —NO₂, —N₃,—C(O)OR⁵, —C(O)NR⁵R⁵,—C(S)NR⁵R⁵, —C(NR⁵)NR⁵R⁵, —CHO, —R⁵CO, —SO₂R⁵, —SOR⁵, —PO(OR⁵)₂—PO(OR⁵), —CO₂H, —SO₃H, —PO₃H, halogen and trihalomethyl; each R⁵ isindependently selected from the group consisting of hydrogen, (C₁-C₆)alkyl, (C₅-C₁₀) aryl, 5-10 membered heteroaryl, (C₆-C₁₆) arylalkyl andsix to sixteen membered heteroarylalkyl; and each K is independentlyselected from the group consisting of oxygen, nitrogen, sulfur andphosphorus.
 54. The compound of claim 53 in which each S¹ isindependently a side-chain of a genetically encoded α-amino acid. 55.The compound of claim 53 in which L is:


56. The laspartomycin derivative of claim 5 5 in which each S¹ isindependently a side-chain of a genetically encoded a-amino acid. 57.The laspartomycin derivative of claim 55 in which n is
 0. 58. Thelaspartomycin derivative of claim 57 in which S¹ is —CH₂—CO₂H or apharmaceutically acceptable salt or hydrate thereof.
 59. Thelaspartomycin derivative of claim 58 in which (X²—X³) taken together are—CONH—.
 60. The laspartomycin derivative of claim 59 in which Y² istetradecan-1-yl.
 61. The laspartomycin derivative of claim 55 in which Lis:

or a salt or hydrate thereof, wherein S² and S³ are each independently aside chain of a genetically encoded a-amino acid.
 62. The laspartomycinderivative of claim 61 in which S² is —CH₂-indol-2-yl and S³ is—CH₂—CO₂H or a pharmaceutically acceptable salt or hydrate thereof. 63.The laspartomycin derivative of claim 62 in which (X²—X³) taken togetherare —CONH—.
 64. The laspartomycin derivative of claim 63 in which Y² isnonan-1-yl.
 65. The laspartomycin derivative of claim 61 in which S² ishydrogen and S³ is —CH₂—CO₂H or a salt thereof.
 66. The laspartomycinderivative of claim 65 in which (X²—X³) taken together are —SO₂NH—. 67.The laspartomycin derivative of claim 66 in which Y² is decan-1-yl. 68.The laspartomycin derivative of claim 55 in which L is:

or a salt or hydrate thereof, wherein S², S³ and S⁴ are eachindependently a side chain of a genetically encoded α-amino acid. 69.The laspartomycin derivative of claim 68 in which S² is —CH₂-indol-2-yl,S³ is —CH₂—CO₂H or a salt thereof and S⁴ is —CH₂—CO₂H or a salt thereof.70. The laspartomycin derivative of claim 69 in which (X²—X³) takentogether are —CONH—.
 71. The laspartomycin derivative of claim 70 inwhich Y² is nonan-1-yl.
 72. A pharmaceutical composition comprising acompound according to claim 48 and a pharmaceutically acceptableexcipient, carrier or diluent.
 73. A method for treating a microbialinfection, said method comprising the step of administering to a subjectan effective amount of a compound according to claim
 49. 74. A methodfor treating a microbial infection, said method comprising the step ofadministering to a subject an effective amount of a compound accordingto claim
 71. 75. A method of inhibiting microbial growth, said methodcomprising the step of administering to a microbe an effective amount ofa compound according to claim
 48. 76. A pharmaceutical compositioncomprising a compound according to claim 49 and a pharmaceuticallyacceptable excipient, carrier or diluent.
 77. A method for treating amicrobial infection, said method comprising the step of administering toa subject an effective amount of a compound according to claim
 49. 78. Amethod for treating a microbial infection, said method comprising thestep of administering to a subject an effective amount of a compoundaccording to claim
 75. 79. A method of inhibiting microbial growth, saidmethod comprising the step of administering to a microbe an effectiveamount of a compound according to claim 49.